System and Method for Detection of Nucleic Acids

ABSTRACT

Embodiments provide detection systems and methods for detecting the presence of a nucleic acid in one or more samples. In a detection method, a sample and one or more nucleic acid probes are introduced into a channel. A first potential difference is applied across the length of the channel in a first direction, and a first electrical property value is detected. Subsequently, a second potential difference is applied across the length of the channel in a second opposite direction, and a second electrical property value is detected. Presence or absence of a nucleic acid in the channel is determined based on a comparison between the first and second electrical property values.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to the following U.S. non-provisionalpatent applications: (i) U.S. patent application Ser. No. 14/507,825,filed on Oct. 6, 2014, titled “System for Detection of Analytes,” (ii)U.S. patent application Ser. No. 14/507,828, filed on Oct. 6, 2014,titled “Method for Detection of Analytes,” (iii) U.S. patent applicationSer. No. 14/507,818, filed on Oct. 6, 2014, titled “System and Methodfor Detection of Mercury,” and (iv) U.S. patent application Ser. No.14/507,820, filed on Oct. 6, 2014, titled “System and Method forDetection of Silver.” The entire contents of each of theabove-referenced patent applications are expressly incorporated hereinby reference.

BACKGROUND

Sensitive and selective detection of chemical and biological analyteshas important implications for medical and environmental testing andresearch. Hospitals and laboratories, for example, routinely testbiological samples to detect potentially toxic substances, such asmercury and silver, in heavy metal poisoning diagnosis. Similarly,measurement of biomolecules, such as nucleic acids, is a foundation ofmodern medicine and is used in medical research, diagnostics, therapyand drug development.

Nanopore sequencing technology is a conventional method of detectingnucleic acid molecules. The concept of nanopore sequencing utilizes ananopore aperture, which is a small hole or pore that extendstransversely through a lipid bilayer membrane, i.e., through the depthor thickness dimension of the membrane. Nanopore sequencing involvescausing a nucleotide to travel through a nanopore in the membrane, i.e.,to travel between the top surface and the bottom surface of the membranealong the depth or thickness dimension of the membrane. A potentialdifference may be applied across the depth or thickness dimension of themembrane to force the nucleotide to travel through the nanopore.Physical changes in the environment of the nucleotide (for example,electric current passing through the nanopore) are detected as thenucleotide traverses through the nanopore. Based on the detected changesin the electrical current, the nucleotide may be identified andsequenced.

Areas for improving and broadening the scope of conventional systems andtechniques of nucleic acid detection have been identified, and technicalsolutions have been implemented in exemplary embodiments.

SUMMARY

In accordance with one exemplary embodiment, a method is provided fordetecting the presence or absence of a nucleic acid in a sample. Themethod includes introducing a sample into a channel, the channel havinga length and a width, the length substantially greater than the width;measuring an electrical property value of an electrical property alongat least a portion of the length of the channel after the sample isintroduced into the channel; accessing a reference electrical propertyvalue, the reference electrical property value associated with theelectrical property of the channel along at least a portion of thelength of the channel prior to introduction of the sample into thechannel; comparing the measured electrical property value and thereference electrical property value; and determining whether the nucleicacid is present in the channel based on the comparison between themeasured electrical property value and the reference electrical propertyvalue.

In accordance with another exemplary embodiment, a method is providedfor detecting the presence or absence of a nucleic acid in a sample. Themethod includes measuring one or more electrical properties of a channelalong at least a portion of the length of the channel, the channelhaving a length and a width, the length substantially greater than thewidth; determining a reference channel electrical property value basedon the one or more electrical properties of the channel measured duringthe previous measuring step; introducing a sample into the channel;measuring the one or more electrical properties of the channel along thesame portion of the length of the channel that was measured in the firstmeasuring step with the sample in the channel; determining a samplechannel electrical property value based on the one or more electricalproperties of the channel measured with the sample in the channel;determining any differences between the sample channel electricalproperty value and the reference channel electrical property value; anddetermining whether a nucleic acid is present in the channel based onthe differences, if any, between the sample channel electrical propertyvalue and the reference channel electrical property value.

In accordance with another exemplary embodiment, a method is providedfor detecting the presence or absence of a nucleic acid in a sample. Themethod includes introducing a sample and one or more nucleic acid probesinto a channel, the channel having a length and a width, the lengthsubstantially greater than the width; measuring an electrical propertyvalue along at least a portion of the length of the channel after thesample and the nucleic acid probes are introduced into the channel;accessing a reference electrical property value from memory, thereference electrical property value associated with at least a portionof the length of the channel; determining any differences between themeasured electrical property value and the reference electrical propertyvalue; and determining whether the nucleic acid probe is present in thechannel based on the differences, if any, between the measuredelectrical property value and the reference electrical property value.

In accordance with another exemplary embodiment, a method is providedfor detecting the presence or absence of a nucleic acid probe in asample. The method includes introducing one or more nucleic acid probesinto a channel, the channel having a length and a width, the lengthbeing substantially greater than the width; measuring one or moreelectrical properties of the channel along at least a portion of thelength of the channel; determining a reference channel electricalproperty value based on the one or more electrical properties of thechannel measured during the previous measuring step; introducing asample into the channel; measuring the one or more electrical propertiesof the channel along at least the portion of the length of the channelafter the sample and the one or more nucleic acid probes are introducedinto the channel; determining an electrical property value based on theone or more electrical properties measured after the one or more nucleicacid probes and the sample are introduced into the channel; determiningany differences between the reference channel electrical property valueand the electrical property value; and determining whether the nucleicacid is present in the channel based on the differences, if any, betweenthe reference channel electrical property value and the electricalproperty value.

In accordance with another exemplary embodiment, a method is providedfor detecting the presence or absence of a nucleic acid in a sample. Themethod includes introducing one or more nucleic acid probes into achannel, the channel having a length and a width, the length beingsubstantially greater than the width; introducing a sample into thechannel; measuring one or more electrical properties of the channelalong at least a portion of the length of the channel after the sampleand the one or more nucleic acid probes are introduced into the channel;determining an electrical property value based on the one or moreelectrical properties measured after the one or more nucleic acid probesand the sample are introduced into the channel; accessing a referencechannel electrical property value, the reference channel electricalproperty value measured prior to introduction of both the one or morenucleic acid probes and the sample into the channel; determining anydifferences between the reference channel electrical property value andthe electrical property value; and determining whether the nucleic acidis present in the channel based on the differences, if any, between thereference channel electrical property value and the electrical propertyvalue.

In accordance with another exemplary embodiment, a method is providedfor detecting the presence or absence of a nucleic acid in a sample. Themethod includes introducing a sample into a channel, the channel havinga length and a width, the length being substantially greater than thewidth; measuring one or more electrical properties of the channel alongat least a portion of the length of the channel; determining a referencechannel electrical property value based on the one or more electricalproperties of the channel measured during the previous measuring step;introducing one or more nucleic acid probes into the channel; measuringthe one or more electrical properties of the channel along at least theportion of the length of the channel after the sample and the one ormore nucleic acid probes are introduced into the channel; determining anelectrical property value based on the one or more electrical propertiesmeasured after the one or more nucleic acid probes and the sample areintroduced into the channel; determining any differences between thereference channel electrical property value and the electrical propertyvalue; and determining whether the nucleic acid is present in thechannel based on the differences, if any, between the reference channelelectrical property value and the electrical property value.

In accordance with another exemplary embodiment, a method is providedfor detecting the presence or absence of a nucleic acid in a sample. Themethod includes introducing a sample into a channel, the channel havinga length and a width, the length being substantially greater than thewidth; introducing one or more nucleic acid probes into the channel;measuring one or more electrical properties of the channel along atleast a portion of the length of the channel after the sample and theone or more nucleic acid probes are introduced into the channel;determining an electrical property value based on the one or moreelectrical properties measured after the one or more nucleic acid probesand the sample are introduced into the channel; accessing a referencechannel electrical property value, the reference channel electricalproperty value measured prior to introduction of both the one or morenucleic acid probes and the sample into the channel; determining anydifferences between the reference channel electrical property value andthe electrical property value; and determining whether the nucleic acidis present in the channel based on the differences, if any, between thereference channel electrical property value and the electrical propertyvalue.

In accordance with another exemplary embodiment, a method is providedfor detecting the presence or absence of a nucleic acid in a sample. Themethod includes coating at least a portion of an inner surface of achannel with one or more nucleic acid probes, the channel having alength and a width, the length substantially greater than the width;measuring one or more electrical properties of the channel along atleast a portion of the length of the channel after the channel is coatedwith the one or more nucleic acid probes; determining a referencechannel electrical property value based on the one or more electricalproperties of the channel measured during the previous measuring step;and storing the reference channel electrical property value for use indetermining whether or not the nucleic acid is present in a sampleintroduced in the channel.

In accordance with another exemplary embodiment, a method is providedfor detecting the presence or absence of a nucleic acid in a sample. Themethod includes introducing a sample and one or more nucleic acid probesinto a channel, the channel having a length and a width, the lengthsubstantially greater than the width. The method also includes applyinga first potential difference across the length of the channel in a firstdirection along the length of the channel. The method also includesmeasuring a first electrical property value of an electrical propertyalong at least a portion of the length of the channel while the firstpotential difference is applied. The method also includes applying asecond potential difference across the length of the channel in a seconddirection along the length of the channel, the second direction oppositeto the first direction. The method also includes measuring a secondelectrical property value of the electrical property along at least theportion of the length of the channel while the second potentialdifference is applied. The method also includes comparing the first andsecond electrical property values. The method also includes determiningwhether a nucleic acid is present in the channel based on the comparisonbetween the first and second electrical property values.

In accordance with another exemplary embodiment, a nucleic aciddetection system is provided. The system includes a substrate, thesubstrate having at least one channel, the at least one channel having alength and a width, the length substantially greater than the width; afirst port in fluid communication with a first end section of the atleast one channel; and a second port in fluid communication with asecond end section of the at least one channel. The system also includesa first electrode electrically connected at the first end section of theat least one channel and a second electrode electrically connected atthe second end section of the at least one channel, the first and secondelectrodes electrically connected to their respective first and secondend sections of the at least one channel to form a channel circuit, thechannel circuit having electrical properties and configured such thatwhen an electrically conductive fluid is present in the at least onechannel, the electrically conductive fluid alters the electricalproperties of the channel circuit. The system further includes adetection circuit in electrical communication with the first and secondelectrodes, the detection circuit including a measurement circuit inelectrical communication with the first and second electrode, themeasurement circuit having a measurement circuit output, the measurementcircuit output including one or more values indicative of one or moreelectrical properties of the channel circuit, the detection circuitincluding a memory in electrical communication with the measurementcircuit output and configured to store the one or more values indicativeof the one or more electrical properties of the channel circuitincluding at least a first value of an electrical property of thechannel circuit and a second value of the electrical property of thechannel circuit, the detection circuit further including a comparisoncircuit in electrical communication with the memory and having as inputsthe at least first and second values, the comparison circuit configuredto provide a comparison circuit output based at least in part on the atleast first and/or second values, the comparison circuit outputindicative of whether a nucleic acid is present in the at least onechannel.

In accordance with another exemplary embodiment, a nucleic aciddetection system is provided. The system includes means foraccommodating a fluid flow; means for introducing a fluid at a firstterminal end of the means for accommodating the fluid flow; means foroutputting the fluid at a second terminal end of the means foraccommodating the fluid flow; means for detecting first and secondvalues of an electrical property of the fluid between the first andsecond terminal ends of the means for accommodating the fluid flow; andmeans for determining whether a nucleic acid is present in the fluidbased on a difference between the first and second values of theelectrical property.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects, features, and advantages ofexemplary embodiments will become more apparent and may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings.

FIG. 1A illustrates a top view of an exemplary nucleic acid detectionsystem including a single channel.

FIG. 1B illustrates a cross-sectional side view of the exemplary nucleicacid detection system of FIG. 1A.

FIG. 2 illustrates a schematic cross-sectional side view of the channelof the exemplary nucleic acid detection system of FIG. 1A, showingaggregate particles and an electrical double layer (EDL).

FIG. 3 illustrates a top view of an exemplary nucleic acid detectionsystem including multiple channels.

FIG. 4 illustrates a top view of another exemplary nucleic aciddetection system including multiple channels.

FIG. 5 is a schematic representing exemplary ions in an exemplarydetection system.

FIGS. 6A and 6B are graphs illustrating exemplary conductivity valuesmeasured in a channel at different concentrations of an exemplaryanalyte.

FIGS. 7A, 7B and 8-15 are flowcharts illustrating exemplary methods fordetecting nucleic acid in a sample.

FIG. 16 is a schematic illustrating formation of a nucleic acidaggregate during detection of a nuclei acid.

FIGS. 17A and 17B are flowcharts illustrating another exemplary methodfor detecting nucleic acid in a sample.

FIG. 18 is a block diagram of an exemplary processing or computingdevice that may be used to implement and execute exemplarycomputer-executable methods.

The accompanying drawings are not intended to be drawn to scale.

DETAILED DESCRIPTION

Areas for improving conventional systems and techniques of detection ofnucleic acids and nucleotides have been identified and technicalsolutions have been implemented in exemplary embodiments. Exemplaryembodiments provide nucleic acid detection systems and techniques thatcouple knowledge of nano and microfluidic surface chemistry,electrokinetics and fluid dynamics to provide novel functionalcapabilities. Compared to conventional techniques such as nanoporetechnology, embodiments provide improved dimensional precision andcontrol, resulting in new functionality and enhanced device performance.

Embodiments provide nucleic acid detection systems and methods fordetecting the presence or absence of a nucleic acid in one or moresamples. An exemplary detection system includes at least one channel foraccommodating a sample and one or more sensor compounds (e.g., one ormore nucleic acid probes), the channel having a width and a length thatis significantly greater in dimension than the width. An exemplarydetection system includes a nucleic acid detection circuit programmed orconfigured to detect one or more electrical properties along at least aportion of the length of the channel to determine whether the channelcontains a nucleic acid and/or nucleotide of interest.

In some cases, the sensor compounds (e.g., one or more nucleic acidprobes) may be selected such that direct or indirect interaction amongthe nucleic acid and/or nucleotide of interest (if present in thesample) and particles of the sensor compounds results in formation of anaggregate that alters one or more electrical properties of the channel.In certain cases, an exemplary channel may be configured to have a depthand/or a width that is substantially equal to or smaller than thediameter of a particle of the aggregate formed in the channel due tointeraction between the nucleic acid and particles of a sensor compound(e.g., one or more nucleic acid probes) used to detect the nucleic acid.As such, formation of the aggregate may cause a partial or completeblockage in the flow of conductive particles in the channel, therebydecreasing the electrical current and electrical conductivity along thelength of the channel and increasing the resistivity along the length ofthe channel. A nucleic acid detection circuit may compare thismeasurable change in the electrical properties of the channel uponintroduction of both the sample and all of the sensor compounds into thechannel, relative to a reference value, to determine if the aggregate ispresent in the channel. Based on a determination that the aggregate ispresent in the channel, the nucleic acid detection circuit may determinethat the sample contains a nucleic acid.

In certain other cases, the aggregate particles may be electricallyconductive, and formation of the aggregate particles may enhance anelectrical pathway along at least a portion of the length of thechannel, thereby causing a measurable increase in the electricalconductivity and electrical current measured along the length of thechannel. In these cases, formation of the aggregate may cause ameasurable decrease in the resistivity along the length of the channel.A nucleic acid detection circuit may compare this measurable change inthe electrical properties of the channel upon introduction of both thesample and all of the sensor compounds into the channel, relative to areference value, to determine if the aggregate is present in thechannel. Based on a determination that the aggregate is present in thechannel, the nucleic acid detection circuit may determine that thesample contains a nucleic acid.

Another exemplary technique for detecting a nucleic acid may involvedetection of the presence of a diode-like behavior in the channel thatis caused by the formation of a nucleic acid aggregate in the channel.In the absence of an aggregate, application of a potential differencehaving a substantially similar magnitude (e.g., +500 V) may result in asubstantially same magnitude of an electrical property (e.g., current)detected along the length of the channel regardless of the direction ofapplication of the potential difference or electric field. If thepotential difference is applied across the length of the channel in afirst direction along the length of the channel (e.g., such that thepositive electrode is at an input port at or near a first end of thechannel and such that the negative electrode is at an output port at ornear a second end of the channel), the resulting current may besubstantially equal in magnitude to the resultant current if thepotential difference is applied in the opposite direction (e.g., suchthat the positive electrode is at the output port and such that thenegative electrode is at the input port).

Formation of a nucleic acid aggregate in the channel may cause adiode-like behavior in which reversal of the direction of the appliedpotential difference or electric field causes a change in the electricalproperty detected in the channel. The diode-like behavior causes thedetected electrical current to vary in magnitude with the direction ofthe electric field. When the electric field or potential difference isapplied in the first direction, the magnitude of the electrical currentmay be different in magnitude than when the potential different orelectric field is applied in the opposite direction. Thus, comparisonbetween a first electrical property value (detected when a potentialdifference is applied in a first direction along the channel length) anda second electrical property value (detected when a potential differenceis applied in a second opposite direction along the channel length) mayenable detection of an aggregate, and thereby detection of the nucleicacid in the sample. If the first and second electrical property valuesare substantially equal in magnitude, then it may be determined that thesample does not contain the nucleic acid. On the other hand, if thefirst and second electrical property values are substantially unequal inmagnitude, then it may be determined that the sample contains thenucleic acid. In other words, the sum of the values of the electricalproperty (positive in one direction, negative in the other direction) issubstantially zero in the absence of an aggregate and substantiallynon-zero in the presence of a nucleic acid aggregate.

In contrast to conventional nanopore techniques, exemplary embodimentsinvolve detecting one or more electrical properties along the length ofthe channel, and not across the depth or thickness dimension of thechannel. The channel of exemplary embodiments has a length that issignificantly greater in dimension that its width and is not configuredas an aperture, hole or pore. The exemplary channel thereby allows asample and sensor compounds to flow along the length of the channelbefore the electrical properties are detected, thereby enabling improveddimensional precision and control over the electrical properties.Furthermore, exemplary embodiments are not limited to detection ofnucleotides as in conventional nanopore techniques.

In certain embodiments, one or more properties of the channel other thanelectrical properties may be detected in determining whether a nucleicacid and/or a nucleotide of interest are present in the channel. Theseproperties may be detected using techniques that include, but are notlimited to, acoustic detection, resonance-wise parametric detection,optical detection, spectroscopic detection, fluorescent dyes, and thelike.

I. Definitions of Terms

Certain terms used in connection with exemplary embodiments are definedbelow.

As used herein, the terms “detection system,” “detection method” and“detection technique” encompass systems and methods for detecting ananalyte in a sample by measuring one or more electrical properties alongat least a portion of a length of at least one channel. The analyte maybe a nucleic acid and/or a nucleotide in one embodiment.

As used herein, the term “channel” encompasses a conduit in a detectionsystem that is configured to have a well-defined inner surface and aninner space bounded by the inner surface that is configured toaccommodate a fluid. In some embodiments, the inner surface of thechannel is micro-fabricated and configured to present a smooth surface.An exemplary channel may have the following dimensions: a length, l,measured along its longest dimension (y-axis) and extending along aplane substantially parallel to a substrate of the detection system; awidth, w, measured along an axis (x-axis) perpendicular to its longestdimension and extending substantially along the plane parallel to thesubstrate; and a depth, d, measured along an axis (z-axis) substantiallyperpendicular to the plane parallel to the substrate. An exemplarychannel may have a length that is substantially greater than its widthand its depth. In some cases, exemplary ratios between the length:widthmay include, but are not limited to, about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1, all intermediate ratios, and the like. In certain cases, anexemplary channel may be configured to have a depth and/or a width thatis substantially equal to or smaller than the diameter of an aggregateparticle that may be formed in the channel due to interaction between asensor compound and an analyte of interest.

As used herein, the term “analyte” encompasses a substance whosepresence or absence may be detected using an exemplary detection systemor method. Exemplary analytes that may be detected using exemplaryembodiments may include organic (e.g., biomolecules) or inorganic (e.g.,metal ions) substances. Certain analytes that may be detected usingexemplary embodiments include, but are not limited to, silver, mercury,one or more solvents, one or more nucleic acids, and/or one or morenucleotides.

As used herein, the term “sample” encompasses a test substance that maybe analyzed by an exemplary detection system or method to determinewhether the sample includes an analyte of interest. Exemplary samplesthat may be tested in exemplary embodiments include, but are not limitedto: any fluids, including those derived from biological fluids likesaliva, blood, plasma, urine, stool; soil samples; municipal watersamples; air samples; and the like.

As used herein, the terms “sensor” and “sensor compound” encompass asubstance that interacts, directly or indirectly via one or more othersensor compounds, with an analyte of interest in a sample to causeformation of an aggregate. In an example in which an analyte of interestis a nucleic acid and/or a nucleotide, a suitable sensor compound may beone or more nucleic acid probes (e.g., one or more nucleic acid captureprobes, one or more nucleic acid cross-linking probes, one or morenucleic acid pre-amplification probes, one or more nucleic acid labelextenders, one or more nucleic acid amplification probes, and the like).

As used herein, the term “aggregate” encompasses a macromolecularstructure composed of particles of an analyte and particles of one ormore sensor compounds. As such, an aggregate particle has a unitdimension or unit size that is larger than the unit dimension or unitsize of an analyte particle and that is larger than the dimension orunit size of a sensor compound. An aggregate may form in a channel of anexemplary detection system due to direct and/or indirect interactionbetween the particles of an analyte and the particles of one or moresensor compounds. In exemplary detection systems and methods fordetecting a particular analyte, one or more sensor compounds may beselected such that the sensor compounds interact with the analyte,directly or indirectly via other substances, to result in formation ofan aggregate in a channel. Presence of the aggregate particles in thechannel therefore indicates presence of the analyte in the channel,whereas absence of the aggregate particles in the channel indicatesabsence of the analyte in the channel.

In certain cases in which a potential difference is applied across atleast a portion of the length of the channel, formation of an aggregatemay cause a partial or complete blockage in fluid flow in the channeland may cause a measurable decrease in an electrical conductivity orcurrent along at least a portion of the length of the channel and/or ameasurable increase in the electrical resistivity. In certain othercases, particles of an aggregate may be electrically conductive, andtherefore formation of the aggregate may enhance the electricalconductivity of the channel, thereby causing a measurable increase inthe electrical conductivity or current along at least a portion of thelength of the channel and/or a measurable decrease in the electricalresistivity.

As used herein, the term “electrical property” encompasses one or morecharacteristics of a channel including, but not limited to, measuresthat quantify how much electric current is conducted along the channel,the ability of the channel (and/or any contents of the channel) toconduct an electric current, how strongly the channel (and/or anycontents of the channel) opposes the flow of electrical current, and thelike. In exemplary embodiments, an electrical property may be detectedalong at least a portion of the length of the channel. Exemplaryelectrical properties detected in embodiments include, but are notlimited to, a measure of an electrical current conducted along at leasta portion of the length of the channel, a measure of an electricalconductivity along at least a portion of the length of the channel, ameasure of electrical resistivity along at least a portion of the lengthof the channel, a measure of potential difference across at least aportion of the length of a channel, combinations thereof, and the like.

As used herein, the term “reference” with respect to an electricalproperty value encompasses a value or range of values of an electricalproperty of a channel prior to a state in which both a sample and allnecessary sensor compounds (e.g., nucleic acid probes) have beenintroduced into the channel and allowed to interact with each other inthe channel. That is, the reference value is a value characterizing thechannel prior to interaction between an analyte of interest in thesample and all of the sensor compounds used to detect the analyte ofinterest. In some cases, the reference value may be detected at a timeperiod after introduction of one or more sensor compounds into thechannel but before introduction of a sample into the channel. In somecases, the reference value may be detected at a time period afterintroduction of the sample into the channel but before introduction ofall of the sensor compounds into the channel (i.e., before introductionof at least one sensor compound into the channel). In some cases, thereference value may be detected at a time period before introduction ofeither the sample or the sensor compounds into the channel. In somecases, the reference value may be detected at a time period beforeintroduction of either the sample or the sensor compounds into thechannel but after introduction of a buffer solution into the channel.

In some cases, the reference value may be predetermined and stored on anon-transitory storage medium from which it may be accessed. In othercases, the reference value may be determined from one or more electricalproperty measurements during use of the detection system.

As used herein, the terms “data,” “content,” “information,” and similarterms may be used interchangeably to refer to data capable of beingtransmitted, received, and/or stored in accordance with embodiments ofthe present invention. Thus, use of any such terms should not be takento limit the spirit and scope of embodiments of the present invention.Further, where a module, processor or device is described herein toreceive data from another module, processor or device, it will beappreciated that the data may be received directly from the anothermodule, processor or device or may be received indirectly via one ormore intermediary modules or devices, such as, for example, one or moreservers, relays, routers, network access points, base stations, hosts,and/or the like, sometimes referred to herein as a “network.” Similarly,where a computing device is described herein to send data to anothercomputing device, it will be appreciated that the data may be sentdirectly to the another computing device or may be sent indirectly viaone or more intermediary computing devices, such as, for example, one ormore servers, relays, routers, network access points, base stations,hosts, and/or the like.

As used herein, the term “module,” encompasses hardware, software and/orfirmware configured to perform one or more particular functions.

As used herein, the term “computer-readable medium” refers to anon-transitory storage hardware, non-transitory storage device ornon-transitory computer system memory that may be accessed by acontroller, a microcontroller, a computational system or a module of acomputational system to encode thereon computer-executable instructionsor software programs.

A “non-transitory computer-readable medium” may be accessed by acomputational system or a module of a computational system to retrieveand/or execute the computer-executable instructions or software programsencoded on the medium. A non-transitory computer-readable medium mayinclude, but is not limited to, one or more types of non-transitoryhardware memory, non-transitory tangible media (for example, one or moremagnetic storage disks, one or more optical disks, one or more USB flashdrives), computer system memory or random access memory (such as, DRAM,SRAM, EDO RAM), and the like.

As used herein, the term “set” refers to a collection of one or moreitems.

As used herein, the term “plurality” refers to two or more items.

As used herein, the terms “equal” and “substantially equal” referinterchangeably, in a broad lay sense, to exact equality or approximateequality within some tolerance.

As used herein, the terms “similar” and “substantially similar” referinterchangeably, in a broad lay sense, to exact sameness or approximatesimilarity within some tolerance.

As used herein, the terms “couple” and “connect” encompass direct orindirect connection among two or more components. For example, a firstcomponent may be coupled to a second component directly or through oneor more intermediate components.

Some exemplary embodiments of the present invention will now bedescribed more fully hereinafter with reference to the accompanyingdrawings in which some, but not all, embodiments of the inventions areshown. Indeed, these inventions may be embodied in many different formsand should not be construed as limited to the embodiments set forthherein; rather, these embodiments are provided so that this disclosurewill satisfy applicable legal requirements. Like numbers refer to likeelements throughout.

II. Exemplary Nucleic Acid Detection Systems

An exemplary nucleic acid detection system includes at least onechannel, and detects one or more electrical properties along at least aportion of the length of the channel to determine whether the channelcontains a particular nucleic acid of interest and/or a particularnucleotide of interest. An exemplary detection system may be configuredto include one or more channels for accommodating a sample and one ormore sensor compounds (e.g., one or more nucleic acid probes), one ormore input ports for introduction of the sample and the sensor compoundsinto the channel and, in some embodiments, one or more output portsthrough which the contents of the channel may be removed.

One or more sensor compounds (e.g., one or more nucleic acid probes) maybe selected such that direct or indirect interaction among the nucleicacid and/or nucleotide of interest (if present in the sample) andparticles of the sensor compounds results in formation of an aggregatethat alters one or more electrical properties of at least a portion ofthe length of the channel. In certain cases, formation of the aggregateparticles may inhibit or block fluid flow in the channel, and maytherefore cause a measurable drop in the electrical conductivity andelectrical current measured along the length of the channel. Similarly,in these cases, formation of the aggregate may cause a measurableincrease in the resistivity along the length of the channel. In certainother cases, the aggregate particles may be electrically conductive, andformation of the aggregate particles may enhance an electrical pathwayalong at least a portion of the length of the channel, thereby causing ameasurable increase in the electrical conductivity and electricalcurrent measured along the length of the channel. In these cases,formation of the aggregate may cause a measurable decrease in theresistivity along the length of the channel.

An exemplary channel may have the following dimensions: a lengthmeasured along its longest dimension (y-axis) and extending along aplane parallel to the substrate of the detection system; a widthmeasured along an axis (x-axis) perpendicular to its longest dimensionand extending along the plane parallel to the substrate; and a depthmeasured along an axis (z-axis) perpendicular to the plane parallel tothe substrate. An exemplary channel may have a length that issubstantially greater than its width and its depth. In some cases,exemplary ratios between the length:width may be about 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, intermediate ratios, and the like.

In certain cases, an exemplary channel may be configured to have a depthand/or a width that is substantially equal to or smaller than thediameter of a particle of an aggregate formed in the channel due tointeraction between the nucleic acid of interest and particles of thesensor compounds (e.g., one or more nucleic acid probes) used to detectthe nucleic acid of interest.

An exemplary channel may have a width taken along the x-axis rangingfrom about 1 nm to about 50,000 nm, but is not limited to this exemplaryrange. An exemplary channel may have a length taken along the y-axisranging from about 10 nm to about 2 cm, but is not limited to thisexemplary range. An exemplary channel may have a depth taken along thez-axis ranging from about 1 nm to about 1 micron, but is not limited tothis exemplary range.

An exemplary channel may have any suitable transverse cross-sectionalshape (i.e., a cross-section taken along the x-z plane) including, butnot limited to, circular, elliptical, rectangular, square, D-shaped (dueto isotropic etching), and the like.

FIGS. 1A and 1B illustrate an exemplary detection system 100 that may beused to detect presence or absence of a particular nucleic acid and/or aparticular nucleotide in a sample.

FIG. 1A is a top view of the system, while FIG. 1B is a cross-sectionalside view of the system. The detection system 100 includes a substrate102 that extends substantially along a horizontal x-y plane. In someembodiments, the substrate 102 may be formed of a dielectric material,for example, silica. Other exemplary materials for the substrate 102include, but are not limited to, glass, sapphire, diamond, and the like.

The substrate 102 may support or include a channel 104 having at leastan inner surface 106 and an inner space 108 for accommodating a fluid.In some cases, the channel 104 may be etched in a top surface of thesubstrate 102. Exemplary materials for the inner surfaces 106 of thechannel 104 include, but are not limited to, glass, silica, and thelike.

The channel 104 and the substrate 102 may be formed of glass in certainembodiments. Biological conditions represent a barrier to the use ofglass-derived implantations due to the slow dissolution of glass intobiological fluids and adhesion of proteins and small molecules to theglass surface. In certain non-limiting embodiments, surface modificationusing a self-assembled monolayer offers an approach for modifying glasssurfaces for nucleic acid detection and analysis. In certainembodiments, at least a portion of the inner surface 106 of the channel104 may be pre-treated or covalently modified to include or be coatedwith a material that enables specific covalent binding of a sensorcompound to the inner surface. In certain embodiments, a cover slip 114covering the channel may also be covalently modified with a material.

Exemplary materials that may be used to modify the inner surface 106 ofthe channel 104 include, but are not limited to, a silane compound(e.g., tricholorsilane, alkylsilane, triethoxysilane, perfluoro silane),zwitterionic sultone, poly(6-9)ethylene glycol (Peg), perfluorooctyl,fluorescein, an aldehyde, a graphene compound, and the like. Thecovalent modification of the inner surface of the channel may preventnon-specific absorption of certain molecules. In one example, covalentmodification of the inner surface may enable covalent bonding of sensorcompound molecules to the inner surface while preventing non-specificabsorption of other molecules to the inner surface. For example,poly(ethylene glycol) (Peg) may be used to modify the inner surface 106of the channel 104 to reduce non-specific adsorption of materials to theinner surface.

In some embodiments, the channel 104 may be nano or micro-fabricated tohave a well-defined and smooth inner surface 106. Exemplary techniquesfor fabricating a channel and modifying the inner surface of a channelare taught in Sumita Pennathur and Pete Crisalli (2014), “LowTemperature Fabrication and Surface Modification Methods for FusedSilica Micro- and Nanochannels,” MRS Proceedings, 1659, pp 15-26.doi:10.1557/op1.2014.32, the entire contents of which are expresslyincorporated herein by reference.

A first end section of the channel 104 may include or be in fluidcommunication with an input port 110, and a second end section of thechannel 104 may include or be in fluid communication with an output port112. In certain non-limiting embodiments, the ports 110 and 112 may beprovided at terminal ends of the channel 104.

The top surface of the substrate 102 having the channel 104 and theports 110, 112 may be covered and sealed with a cover slip 114 in someembodiments.

A first electrode 116 may be electrically connected at the first endsection of the channel 104, for example, at or near the input port 110.A second electrode 118 may be electrically connected at the second endsection of the channel 104, for example, at or near the output port 112.The first and second electrodes 116, 118 may be electrically connectedto a power supply or voltage source 120 in order to apply a potentialdifference between the first and second electrodes. That is, thepotential difference is applied across at least a portion of the lengthof the channel. When a fluid is present in the channel 104 and is underthe influence of the applied potential difference, the electrodes 116,118 and the fluid may create a complete electrical pathway.

The power supply or voltage source 120 may be configured to apply anelectric field in a reversible manner such that a potential differenceis applied in a first direction along the channel length (along they-axis) and also in a second opposite direction (along the y-axis). Inone example in which the electric field or potential differencedirection is in a first direction, the positive electrode may beconnected at the first end section of the channel 104, for example, ator near the input port 110, and the negative electrode may be connectedat the second end section of the channel 104, for example, at or nearthe output port 112. In another example in which the electric field orpotential difference direction is in a second opposite direction, thenegative electrode may be connected at the first end section of thechannel 104, for example, at or near the input port 110, and thepositive electrode may be connected at the second end section of thechannel 104, for example, at or near the output port 112.

The first and second end sections of the channel 104 (i.e., at or nearthe input port 110 and the output port 112) may be electricallyconnected to a nucleic acid detection circuit 122 that is programmed orconfigured to detect values of one or more electrical properties of thechannel 104 for determining whether the particular nucleic acid and/ornucleotide is present or absent in the channel 104. The electricalproperty values may be detected at a single time period (for example, acertain time period after introduction of a sample and one or moresensor compounds into the channel), or at multiple different timeperiods (for example, before and after introduction of both the sampleand one or more sensor compound into the channel). Exemplary electricalproperties detected may include, but are not limited to, electricalcurrent, conductivity voltage, resistance, and the like. Certainexemplary nucleic acid detection circuits 122 may include or beconfigured as a processor or a computing device, for example as device1700 illustrated in FIG. 18. Certain other nucleic acid detectioncircuits 122 may include, but are not limited to, an ammeter, avoltmeter, an ohmmeter, and the like.

In one embodiment, the nucleic acid detection circuit 122 may include ameasurement circuit 123 programmed or configured to measure one or moreelectrical property values along at least a portion of a length of thechannel 104. The nucleic acid detection circuit 122 may also include anequilibration circuit 124 that is programmed or configured toperiodically or continually monitor one or more values of an electricalproperty of the channel over a time period, and to select a single oneof the values after the values have reached equilibrium (i.e., havestopped varying beyond a certain threshold of variance or tolerance).

The nucleic acid detection circuit 122 may also include a comparisoncircuit 126 that is programmed or configured to compare two or moreelectrical property values of the channel, for example, a referenceelectrical property value (measured before a state in which both thesample and all of the sensor compounds have been introduced into thechannel) and an electrical property value (measured after introductionof the sample and all of the sensor compound into the channel). Thecomparison circuit 126 may use the comparison in order to determinewhether the nucleic acid is present or absent in the channel. In oneembodiment, the comparison circuit 126 may calculate a differencebetween the measured electrical property value and the referenceelectrical property value, and compare the difference to a predeterminedvalue indicative of the presence or absence of the nucleic acid in thechannel.

In certain embodiments, upon introduction of both the sample and thesensor compound into the channel, the comparison circuit 126 may beprogrammed or configured to compare a first electrical property value(e.g., magnitude of electrical current) when the electric field orpotential difference is applied across the channel in a first directionalong the length of the channel to a second electrical property value(e.g., magnitude of electrical current) when the electric field orpotential difference is applied across the channel in a second oppositedirection along the length of the channel. In one embodiment, thecomparison circuit 126 may calculate a difference between the magnitudesof the first and second values, and compare the difference to apredetermined value (e.g., whether the difference is substantially zero)indicative of the presence or absence of a nucleic acid in the channel.For example, if the difference is substantially zero, this indicatesabsence of a nucleic acid aggregate in the channel, i.e., absence of thenucleic acid in the channel. If the difference is substantiallynon-zero, this indicates presence of a nucleic acid aggregate in thechannel, i.e., presence of the nucleic acid in the channel.

In certain embodiments, the nucleic acid detection circuit 122 may beprogrammed or configured to determine an absolute concentration of thenucleic acid in a sample, and/or a relative concentration of the nucleicacid relative to one or more additional substances in a sample.

In some embodiments, the comparison circuit 124 and the equilibrationcircuit 126 may be configured as separate circuits or modules, while inother embodiments, the may be configured as a single integrated circuitor module.

The nucleic acid detection circuit 122 may have an output 128 that may,in some embodiments, be connected to one or more external devices ormodules. For example, the nucleic acid detection circuit 122 maytransmit a reference electrical property value and/or one or moremeasured electrical property values to one or more of: a processor 130for further computation, processing and analysis, a non-transitorystorage device or memory 132 for storage of the values, and a visualdisplay device 134 for display of the values to a user. In some cases,the nucleic acid detection circuit 122 may itself generate an indicationof whether the sample includes the nucleic acid, and may transmit thisindication to the processor 130, the non-transitory storage device ormemory 132 and/or the visual display device 134.

In an exemplary method of using the system of FIGS. 1A and 1B, one ormore sensor compounds (e.g., one or more nucleic acid probes) and asample may be sequentially or concurrently introduced into the channel.

When flow of the fluid and/or flow of the charged particles in the fluidis uninhibited (for example, due to absence of an aggregate), theconductive particles or ions in the fluid may travel along at least aportion of the length of the channel 104 along the y-axis from the inputport 110 toward the output port 112. The movement of the conductiveparticles or ions may result in a first or “reference” electricalproperty value or range of values (e.g., of an electrical current,conductivity, resistivity) being detected by the nucleic acid detectioncircuit 122 along at least a portion of the length of the channel 104.In some embodiments, the equilibration circuit 124 may periodically orcontinually monitor electrical property values during a time perioduntil the values reach equilibrium. The equilibration circuit 124 maythen select one of the values as the reference electrical property valueto avoid the influence of transient changes in the electrical property.

The term “reference” electrical property value may refer to a value orrange of values of an electrical property of a channel prior tointroduction of a sample and all of the sensor compounds (e.g., one ormore nucleic acid probes) into the channel. That is, the reference valueis a value characterizing the channel prior to any interaction betweenthe nucleic acid in the sample and all of the sensor compounds. In somecases, the reference value may be detected at a time period afterintroduction of a sensor compound into the channel but beforeintroduction of the sample and additional sensor compounds into thechannel. In some cases, the reference value may be detected at a timeperiod after introduction of a sensor compound and the sample into thechannel but before introduction of additional sensor compounds into thechannel. In some cases, the reference value may be detected at a timeperiod before introduction of the sample or the sensor compounds intothe channel. In some cases, the reference value may be predetermined andstored on a non-transitory storage medium from which it may be accessed.

In some cases, formation of an electrically conductive aggregate in thechannel (due to interactions between a nucleic acid of interest in thesample and one or more nucleic acid probes) may enhance the electricalpathway along at least a portion of the length of the channel 104. Inthis case, the nucleic acid detection circuit 122 may detect a secondelectrical property value or range of values (e.g., of an electricalcurrent, conductivity, resistivity) along at least a portion of thelength of the channel 104. In some embodiments, the nucleic aciddetection circuit 122 may wait for a waiting or adjustment time periodafter introduction of the sample and all of the sensor compounds intothe channel prior to detecting the second electrical property value. Thewaiting or adjustment time period allows an aggregate to form in thechannel and for the aggregate formation to alter the electricalproperties of the channel.

In some embodiments, the equilibration circuit 124 may periodically orcontinually monitor electrical property values during a time periodafter the introduction of the sample and all of the sensor compoundsuntil the values reach equilibrium. The equilibration circuit 124 maythen select one of the values as the second electrical property value toavoid the influence of transient changes in the electrical property.

The comparison circuit 126 may compare the second electrical propertyvalue to the reference electrical property value. If it is determinedthat the difference between the second value and the reference valuecorresponds to a predetermined range of increase in current orconductivity (or decrease in resistivity), the nucleic acid detectioncircuit 122 may determine that an aggregate is present in the channeland that, therefore, the nucleic acid is present in the sample.

In certain other cases, when flow of the fluid in the channel and/orflow of the charged particles in the fluid is partially or completelyblocked (for example, by formation of an aggregate), the conductiveparticles or ions in the fluid may be unable to freely travel along atleast a portion of the length of the channel 104 along the y-axis fromthe input port 110 toward the output port 112. The hindered or stoppedmovement of the conductive particles or ions may result in a thirdelectrical property value or range of values (e.g., of an electricalcurrent, conductivity, resistivity) being detected by the nucleic aciddetection circuit 122 along at least a portion of the length of thechannel 104. The third electrical property value may be detected inaddition to or instead of the second electrical property value. In someembodiments, the nucleic acid detection circuit 122 may wait for awaiting or adjustment time period after introduction of both the sampleand all of the sensor compounds into the channel prior to detecting thethird electrical property value. The waiting or adjustment time periodallows an aggregate to form in the channel and for the aggregateformation to alter the electrical properties of the channel.

In some embodiments, the equilibration circuit 124 may periodically orcontinually monitor electrical property values during a time periodafter the introduction of the sample and all of the sensor compoundsuntil the values reach equilibrium. The equilibration circuit 124 maythen select one of the values as the third electrical property value toavoid the influence of transient changes in the electrical property.

The comparison circuit 126 may compare the third electrical propertyvalue to the reference electrical property value. If it is determinedthat the difference between the third value and the reference valuecorresponds to a predetermined range of decrease in current orconductivity (or increase in resistivity), the nucleic acid detectioncircuit 122 may determine that an aggregate is present in the channeland that, therefore, the nucleic acid is present in the sample.

The fluid flow along the length of the channel may depend on the size ofthe aggregate particles in relation to the dimensions of the channel,and the formation of an electrical double layer (EDL) at the innersurface of the channel. FIG. 2 illustrates a cross-sectional side viewof an exemplary channel of the detection system of FIGS. 1A and 1B, inwhich the combination of an electric double layer (EDL) 202 at the innersurface of the channel and aggregate particles 204 is shown to inhibitfluid flow in the channel.

In general terms, an EDL is a region of net charge between a chargedsolid (e.g., the inner surface of the channel, an analyte particle, anaggregate particle) and an electrolyte-containing solution (e.g., thefluid contents of the channel). EDLs exist around both the inner surfaceof the channel and around any nucleic acid particles and aggregateparticles within the channel. The counter-ions from the electrolyte areattracted towards the charge of the inner surface of the channel, andinduce a region of net charge. The EDL affects ion flow within thechannel and around analyte particles and aggregate particles ofinterest, creating a diode-like behavior by not allowing any of thecounter-ions to pass through the length of the channel.

To mathematically solve for the characteristic length of the EDL, thePoisson-Boltzmann (PB) equation and/or Poisson-Nernst-Plank equations(PNP) may be solved. These solutions are coupled to the Navier-Stokes(NS) equations for fluid flow to create a nonlinear set of coupledequations that are analyzed to understand the operation of the exemplarysystem.

In view of the dimensional interplay among the channel surface, the EDLsand the aggregate particles, exemplary channels may be configured andconstructed with carefully selected dimensional parameters that ensurethat flow of conductive ions is substantially inhibited along the lengthof the channel when an aggregate of a certain predetermined size isformed in the channel. In certain cases, an exemplary channel may beconfigured to have a depth and/or a width that is substantially equal toor smaller than the diameter of an aggregate particle formed in thechannel during nucleic acid detection. In certain embodiments, the sizesof the EDLs may also be taken into account in selecting dimensionalparameters for the channel. In certain cases, an exemplary channel maybe configured to have a depth and/or a width that is substantially equalto or smaller than the dimension of the EDL generated around the innersurface of the channel and the aggregate particles in the channel.

In certain embodiments, prior to use of the detection system, thechannel may be free of the sensor compounds (e.g., one or more nucleicacid probes). That is, a manufacturer of the detection system may notpre-treat or modify the channel to include the sensor compound. In thiscase, during use, a user may introduce one or more sensor compounds, forexample in an electrolyte buffer, into the channel and detect areference electrical property value of the channel with the sensorcompound but in the absence of a sample.

In certain other embodiments, prior to use of the detection system, thechannel may be pre-treated or modified so that at least a portion of aninner surface of the channel includes or is coated with a sensorcompound (e.g., one or more nucleic acid capture probes). In oneexample, the manufacturer may detect a reference electrical propertyvalue of the channel modified with the sensor compound and, during use,a user may use the stored reference electrical property value. That is,a manufacturer of the detection system may pre-treat or modify thechannel to include a sensor compound. In this case, a user may need tointroduce the sample and one or more additional sensor compounds intothe channel.

Certain exemplary detection systems may include a single channel.Certain other exemplary detection systems may include multiple channelsprovided on a single substrate. Such detection systems may include anysuitable number of channels including, but not limited to, 2, 3, 4, 5,6, 7, 8, 9, 10, and the like.

In one embodiment, a detection system may include a plurality ofchannels in which at least two channels operate independent of eachother. The exemplary channel 104 and associated components of FIGS. 1Aand 1B may be reproduced on the same substrate to achieve such amulti-channel detection system. The multiple channels may be used todetect the same nucleic acid in the same sample, different nucleic acidsin the same sample, the same nucleic acid in different samples, and/ordifferent nucleic acids in different samples.

In another embodiment, a detection system may include a plurality ofchannels in which at least two channels operate in cooperation with eachother. FIG. 3 illustrates an exemplary detection system 300 including asubstrate 302. The substrate 302 may include a plurality of channels304, 306 that may be used to detect a nucleic acid in the same sample.Although two channels are represented, more channels may be provided inthe detection system. The provision of multiple channels may allowredundancy and error-checking functionalities, whereby differentdetection results in the channels may indicate that the detection systemis not performing reliably and whereby the same result in the channelsmay indicate that the detection system is performing reliably. In theformer case, the detection system may need to be repaired, recalibratedor discarded.

First end sections of the first channel 304 and the second channel 306may include or be in fluid communication with a common input port 308 atwhich a sample and one or more sensor compounds may be introduced intothe detection system. A second end section of the first channel 304 mayinclude or be in fluid communication with a first output port 310, and asecond end section of the second channel 306 may include or be in fluidcommunication with a second output port 312. The output ports 310 and312 may not be in fluid communication with each other.

The detection system 300 may include electrodes 314, 316A and 316B thatmay be electrically connected at or near the end sections of the firstand second channels 304, 306. The electrodes 314, 316A and 316B mayconnect the channels 304, 306 to a voltage or power supply 332 in orderto apply a potential difference across the input port 308 and the firstoutput port 310 and across the input port 308 and the second output port312. Similarly, a nucleic acid detection circuit 318 may be electricallyconnected at or near the end sections of the first and second channels304, 306 to determine whether the sample introduced into both channelscontain a nucleic acid.

Components represented in FIG. 3 that are in common with componentsrepresented in FIGS. 1A and 1B are described in connection with FIGS. 1Aand 1B.

In another embodiment, a detection system may include a plurality ofchannels in which at least two channels operate in cooperation with eachother. FIG. 4 illustrates an exemplary detection system 400 including asubstrate 402. The substrate 402 may include a plurality of channels404, 406 that may be used to detect a nucleic acid in different samplesor different analytes in the same sample. Although two channels arerepresented, more channels may be provided in the detection system. Theprovision of multiple channels may allow concurrent detection ofmultiple nucleic acids in the same sample or the same nucleic acid inmultiple samples, thereby improving the speed and throughput of thedetection system.

First end sections of the first channel 404 and the second channel 406may include or be in fluid communication with a common first input port408 at which a sample or one or more sensor compounds may be introducedinto the detection system. In addition, the first end section of thefirst channel 404 may include or be in fluid communication with a secondinput port 414. The first end section of the second channel 406 mayinclude or be in fluid communication with a third input port 416. Thesecond and third input ports 414, 416 may not be in fluid communicationwith other.

A second end section of the first channel 404 may include or be in fluidcommunication with a first output port 410, and a second end section ofthe second channel 406 may include or be in fluid communication with asecond output port 412. The output ports 410 and 412 may not be in fluidcommunication with each other.

The detection system 400 may include electrodes 418, 420 and 422 thatmay be electrically connected at or near the end sections of the firstand second channels 404, 406. The electrodes may electrically connectthe first and second channels to a voltage or power source 436 in orderto apply a potential difference across the first input port 408 and thefirst output port 410 and across the first input port 408 and the secondoutput port 412. Similarly, a nucleic acid detection circuit 424 may beelectrically connected at or near the end sections of the first andsecond channels 404, 406 to determine whether one or more samplesintroduced into the channels contain a nucleic acid.

Components represented in FIG. 4 that are in common with componentsrepresented in FIGS. 1A and 1B are described in connection with FIGS. 1Aand 1B.

In an exemplary method of using the system 400 of FIG. 4, a sample maybe introduced into the common first input port 408, and first and secondsets of sensor compounds may be introduced at the second and third inputports 414 and 416, respectively. As a result, based on measurementstaken at the first and second end sections of the first channel 404, thenucleic acid detection circuit 424 may determine whether the sampleincludes a first analyte of interest (which interacts with the first setof sensor compounds in the first channel to form an aggregate). Based onmeasurements taken at the first and second end sections of the secondchannel 406, the nucleic acid detection circuit 424 may determinewhether the sample includes a second analyte of interest (whichinteracts with the second set of sensor compounds in the second channelto form an aggregate).

In another exemplary method of use, one or more sensor compounds may beintroduced into the common first input port 408, and first and secondsamples may be introduced at the second and third input ports 414 and416, respectively. As a result, based on measurements taken at the firstand second end sections of the first channel 404, the nucleic aciddetection circuit 424 may determine whether the first sample includes anucleic acid (which interacts with the sensor compounds in the firstchannel to form an aggregate). Based on measurements taken at the firstand second end sections of the second channel 406, the nucleic aciddetection circuit may 424 determine whether the second sample includesthe nucleic acid (which interacts with the sensor compounds in thesecond channel to form an aggregate).

In certain embodiments, the systems illustrated in FIGS. 1A, 1B, 3 and 4may be used to determine an absolute or relative concentration of anucleic acid based on one or more electrical property values of thechannel. The concentration of the nucleic acid may be determined in sucha manner because the channels of exemplary detection systems have a highinner surface area to volume ratio. At low concentrations of the nucleicacid, electrical conductivity in the channel is dominated by surfacecharges. As such, measurements of electrical properties of the channelcan enable distinction between different ions. As a result, unique andsensitive measurements of the bulk flow in the channel can be used todetermine information on the surface charges at the inner surface of thechannel. Exemplary embodiments may thus compute predetermined ranges ofelectrical property values of the channel that are characteristic of theparticles of the nucleic acid ions given the dimensions of the channeland at different concentrations of the nucleic acid. These predeterminedvalues may then be used to determine an unknown concentration of thenucleic acid in a sample.

Detailed information on surface charges in the channel for differentions is presented in the following papers, the entire contents of whichare expressly incorporated herein by reference: “Surface-dependentchemical equilibrium constants and capacitances for bare and3-cyanopropyldimethylchlorosilane coated silica nanochannels,” M. B.Andersen, J. Frey, S. Pennathur and H. Bruus, J. Colloid Interface Sci.353, 301-310 (2011), and “Hydronium-domination ion transport incarbon-dioxide-saturated electrolytes at low salt concentrations innanochannels,” K. L. Jensen, J. T. Kristensen, A. M. Crumrine, M. B.Andersen, H. Bruus and S. Pennathur, Phys. Review E. 83, 5, 056307.

FIG. 5 is a schematic drawing of the inside of a channel including aninner surface of the channel 502, an immobile layer of fluid 504 lyingimmediately adjacent to the inner surface of the channel, a diffusivelayer of fluid 506 lying immediately adjacent to the immobile layer, anda bulk fluid flow layer 508 lying immediately adjacent to the diffusivelayer. Exemplary ions are represented in each of the fluid layers. Uponapplication of a potential difference across the length of the channel,an electrical property value may be detected along at least a portion ofthe length of the channel (for example, by the nucleic acid detectioncircuit 122). The comparison circuit 126 may be used to compare themeasured electrical property value to a predetermined range ofelectrical property values that correspond to a particular concentrationor range of concentration values of a nucleic acid. The concentrationdetermined may be an absolute concentration of the nucleic acid or arelative concentration of the nucleic acid with respect to theconcentrations of one or more other substances in the channel.

FIGS. 6A and 6B are graphs showing conductivity values measured in achannel for different test cases. In each test case, a differentrelative concentration of an analyte relative to concentrations of twoadditional substances (in this case, ammonium and hydrogen peroxide) isused, and the corresponding conductivity value is determined in thechannel. In one embodiment, Standard Clean 1 or SC1 is used a solutionin the test cases. Details of SC1 can be found athttp://en.wikipedia.org/wiki/RCA_clean, the entire contents of which areexpressly incorporated herein by reference. The ratios of concentrationsamong the three substances in the test cases represented in FIGS. 6A and6B are presented in Table 1 below.

TABLE 1 Test case ratios for the concentration of water to theconcentration of hydrogen peroxide to the concentration of ammoniumhydroxide Test Concentration Ratio of Water:Hydrogen CasePeroxide:Ammonium Hydroxide A 5:1:1 B 4.8:1.3:0.75 C 5.1:0.62:1.3 D5.26:0.24:1.5 E 4.92:1.3:0.83 F 3500:10:10 G 3501:3.95:14 H 3497:16:06 I3501:6.97:12 J 3499:12.5:8.3

The lower the concentration of an analyte, the easier it is to measuredifferences in relative concentrations between the analyte and othersubstances. For example, at concentration ratios of about 1000:1:1,detection sensitivity on the order of 1-10 ppm may be achieved in theexemplary detection system. At concentration ratios of about 350:1:1,detection sensitivity on the order of 100 ppm may be achieved. Atconcentration ratios of about 5:1:1, detection sensitivity on the orderof 10,000 ppm may be achieved.

The substrate 102, the channel 104 and the cover slip 114 of FIGS. 1Aand 1B may be formed of glass in certain embodiments. Biologicalconditions represent a barrier to the use of glass-derived implantationsdue to the slow dissolution of glass into biological fluids and adhesionof proteins and small molecules to the glass surface. In exemplaryembodiments, surface modification using a self-assembled monolayeroffers an approach for modifying glass surfaces for nucleic aciddetection and analysis. In certain embodiments, at least a portion ofthe inner surface 106 of the channel 104 and/or the inner surface of thecover slip 114 may be pre-treated or covalently modified to include orbe coated with a material that enables specific covalent binding of asensor compound (e.g., one or more nucleic acid probes) to the innersurface.

Exemplary materials that may be used to modify the inner surface of thechannel and/or the cover slip include, but are not limited to, a silanecompound (e.g., tricholorsilane, alkylsilane, triethoxysilane, perfluorosilane), zwitterionic sultone, poly(6-9)ethylene glycol (Peg),perfluorooctyl, fluorescein, an aldehyde, a graphene compound, and thelike. The covalent modification of the inner surface of the channel mayprevent non-specific absorption of certain molecules. In one example,covalent modification of the inner surface may enable covalent bondingof one or more nucleic acid probes to the inner surface while preventnon-specific absorption of other molecules to the inner surface.

As one example of a modification material, alkylsilanes are a group ofmolecules that form covalent monolayers on the surfaces of silicon andglass. Alkylsilanes have three distinct regions: a head group surroundedby good leaving groups, a long alkyl chai, and a terminal end group. Thehead group, usually containing a halogen, alkoxy or other leaving group,allows the molecule to covalently anchor to the solid glass surfaceunder appropriate reaction conditions. The alkyl chain contributes tothe stability and ordering of the monolayer through Vander-Waalsinteractions, which allows for the assembly of a well ordered monolayer.The terminal end group allows for the functionalization and tunabilityof chemical surface properties by using techniques including, but notlimited to, nucleophilic substitution reactions, click chemistry orpolymerization reactions.

In one exemplary technique of treating the inner surface with a silanecompound, a solution is produced. The solution may be between 0.1% and4% v/v (if silane is liquid) or w/v (if silane is a solid) ofappropriate chloro-, trichloro-, trimethoxy- or triethoxysilane in theappropriate solvent (e.g. toluene for trimethoxy- or triethoxysilanes,ethanol for chloro- or trichlorosilanes or water with a pH between 3.5to 5.5 for trimethoxysilanes). The solution may be filtered through a0.2 micron surfactant free cellulose acetate (SFCA) filter. About 10 μLof the filtered silane solution may be added to a port of the channeland allowed to capillary fill the channel. This may or may not beobserved by light microscopy and may take between five and forty minutesdepending upon the solvent composition. After capillary filling iscomplete, about 10 μL of the filtered silane solution may be added tothe remaining ports of the channel. The entire channel may then beimmersed in the filtered silane solution and allowed to react for adesired amount of time (for example, about 1 to 24 hours) at a desiredtemperature (for example, about 20° C. to 80° C. depending upon thespecific silane and solvent composition used for the modification).After the desired reaction time is over, the silanization process may bequenched using one of the following techniques, and catalytic amount ofacetic acid may be added to toluene or ethanol-based surfacemodifications in some cases.

In one exemplary technique of quenching, the entire channel may betransferred to a container filled with 0.2 micron SFCA filtered ethanol,and stored until the desired time for use or further modification. Inanother exemplary technique of quenching, the channel may beelectrokinetically washed with an appropriate solvent composition. Inone electrokinetic washing technique for toluene modification of achannel, toluene is electrokinetically driven through the channel at a10V to 1000V differential between electrodes for about 5 to 15 minutes,followed by electrokinetically driving ethanol through the channel at a10V to 1000V differential between electrodes for about 5 to 15 minutes,followed by electrokinetically driving a 1:1 mixture of ethanol:waterthrough the channel at 10V to 1000V differential between electrodes forabout 5 to 15 minutes, followed by a final electrokinetic driving ofwater through the channel at 10V to 1000V for about 5 to 15 minutes.Proper operation of the channel may be confirmed by measuring thecurrent at 1000V applied field to an added 50 mM sodium borate buffer inthe channel (giving a current reading of approximately 330 nA based onchannel dimensions) and re-addition of ultrapure (e.g., MilliQultrapure) water at the same applied field affording a current of lessthan about 20 nA.

Table 2 summarizes certain exemplary materials that may be used tomodify an inner surface of a channel and/or an inner surface of a coverslip covering the channel.

TABLE 2 Exemplary materials for modification of channel ModificationStructure Poly(6-9)ethylene glycol (Peg)

Octyldimethyl (ODM)

Octyldimethyl + Peg 100,000 ODM + Poly(ethylene oxide) (average MW100,000) grafted under radical conditions Octyldimethyl + Peg 400,000ODM + Poly(ethylene oxide) (average MW 400,000) grafted under radicalconditions Octyldimethyl + Peg 600,000 ODM + Poly(ethylene oxide)(average MW 600,000) grafted under radical conditions Octyldimethyl +Peg 1,000,000 ODM + Poly(ethylene oxide) (average MW 1,000,000) graftedunder radical conditions Octyldimethyl + PVP 1,300,000 ODM +Polyvinylpyrrolidone (average MW 1,300,000) grafted under radicalconditions 3-(dimethylaminopropyl)

3-(aminopropyl)

Perfluorooctyl

Perfluorodecyl

3-(trifluoromethyl)propyl

3-cyanopropyl

Propylmethacrylate

3-mercaptopropyl

3-mercaptopropyl + Peg 5000 Maleimide

3-mercaptopropyl + acrylamide

3-mercaptopropyl + trimethylammonium

Zwitterionic sultone

Zwitterionic phosphate

III. Exemplary Nucleic Acid Detection Techniques

Exemplary techniques enable detection of particular nucleic acids and/ornucleotides (e.g., DNA, RNA) in a sample using one or more sensorcompounds (i.e., one or more nucleic acid probes). An exemplary nucleicacid that may be detected is glyceraldehyde-3-phosphate dehydrogenase(GAPD) messenger RNA (mRNA) included in a total RNA extract. One or moreexemplary sensor compounds that may be used to test for the presence orabsence of a nucleic acid include one or more nucleic acid probes thatbind, directly or indirectly, with the analyte nucleic acid to form anelectrically conductive aggregate. The analyte nucleic acid and the oneor more nucleic acid probes may interact to form an aggregate that maycoat or cover at least part of the inner surface or the inner space ofthe channel, thereby enhancing an electrical pathway along the length ofthe channel. If the aggregate is electrically conductive, this may causea measurable increase in an electrical current and/or electricalconductivity measured along at least a portion of the length of thechannel, and a measurable decrease in an electrical resistivity measuredalong at least a portion of the length of the channel.

In certain embodiments, the electrodes used in the detection system maybe metallic, for example, aluminum, manganese and platinum. In otherembodiments, the electrodes used in the detection system may benon-metallic.

Exemplary techniques may introduce both the sample and all of the sensorcompounds (e.g., one or more nucleic acid probes) into a channel in thedetection system that is especially configured and dimensioned to allownucleic acid detection. In certain embodiments, the channel may beconfigured so that its depth and/or its width are substantially equal toor lower than a diameter of the aggregate particle. Upon introduction ofthe sample and the sensor compounds into the channel, formation of theaggregate may indicate presence of a nucleic acid in the sample, whileabsence of the aggregate may indicate absence of the nucleic acid in thesample.

When flow of the fluid and/or flow of the charged particles in the fluidis uninhibited (for example, due to absence of an aggregate), theconductive particles or ions in the fluid may travel along at least aportion of the length of the channel along the y-axis from the inputport toward the output port. The movement of the conductive particles orions may result in a first or “reference” electrical property value orrange of values (e.g., of an electrical current, conductivity,resistivity) being detected by a nucleic acid detection circuit along atleast a portion of the length of the channel. In some embodiments, anequilibration circuit may periodically or continually monitor electricalproperty values during a time period until the values reach equilibrium.The equilibration circuit may then select one of the values as thereference electrical property value to avoid the influence of transientchanges in the electrical property.

The term “reference” electrical property value may refer to a value orrange of values of an electrical property of a channel prior tointroduction of a sample and all of the sensor compounds (e.g., one ormore nucleic acid probes) into the channel. That is, the reference valueis a value characterizing the channel prior to any interaction betweenthe nucleic acid in the sample and all of the sensor compounds. In somecases, the reference value may be detected at a time period afterintroduction of a sensor compound into the channel but beforeintroduction of the sample and additional sensor compounds into thechannel. In some cases, the reference value may be detected at a timeperiod after introduction of a sensor compound and the sample into thechannel but before introduction of additional sensor compounds into thechannel. In some cases, the reference value may be detected at a timeperiod before introduction of the sample or the sensor compounds intothe channel. In some cases, the reference value may be predetermined andstored on a non-transitory storage medium from which it may be accessed.

In some cases, formation of an electrically conductive aggregate in thechannel (due to interactions between a nucleic acid of interest in thesample and one or more nucleic acid probes) may enhance the electricalpathway along at least a portion of the length of the channel. In thiscase, the nucleic acid detection circuit may detect a second electricalproperty value or range of values (e.g., of an electrical current,conductivity, resistivity) along at least a portion of the length of thechannel. In some embodiments, the nucleic acid detection circuit maywait for a waiting or adjustment time period after introduction of thesample and all of the sensor compounds into the channel prior todetecting the second electrical property value. The waiting oradjustment time period allows an aggregate to form in the channel andfor the aggregate formation to alter the electrical properties of thechannel.

In some embodiments, the equilibration circuit may periodically orcontinually monitor electrical property values during a time periodafter the introduction of the sample and all of the sensor compoundsuntil the values reach equilibrium. The equilibration circuit may thenselect one of the values as the second electrical property value toavoid the influence of transient changes in the electrical property.

The comparison circuit may compare the second electrical property valueto the reference electrical property value. If it is determined that thedifference between the second value and the reference value correspondsto a predetermined range of increase in current or conductivity (ordecrease in resistivity), the nucleic acid detection circuit maydetermine that an aggregate is present in the channel and that,therefore, the nucleic acid is present in the sample.

In certain other cases, when flow of the fluid in the channel and/orflow of the charged particles in the fluid is partially or completelyblocked (for example, by formation of an aggregate), the conductiveparticles or ions in the fluid may be unable to freely travel along atleast a portion of the length of the channel along the y-axis from theinput port toward the output port. The hindered or stopped movement ofthe conductive particles or ions may result in a third electricalproperty value or range of values (e.g., of an electrical current,conductivity, resistivity) being detected by the nucleic acid detectioncircuit along at least a portion of the length of the channel. The thirdelectrical property value may be detected in addition to or instead ofthe second electrical property value. In some embodiments, the nucleicacid detection circuit may wait for a waiting or adjustment time periodafter introduction of both the sample and all of the sensor compoundsinto the channel prior to detecting the third electrical property value.The waiting or adjustment time period allows an aggregate to form in thechannel and for the aggregate formation to alter the electricalproperties of the channel.

In some embodiments, the equilibration circuit may periodically orcontinually monitor electrical property values during a time periodafter the introduction of the sample and all of the sensor compoundsuntil the values reach equilibrium. The equilibration circuit may thenselect one of the values as the third electrical property value to avoidthe influence of transient changes in the electrical property.

The comparison circuit may compare the third electrical property valueto the reference electrical property value. If it is determined that thedifference between the third value and the reference value correspondsto a predetermined range of decrease in current or conductivity (orincrease in resistivity), the nucleic acid detection circuit maydetermine that an aggregate is present in the channel and that,therefore, the nucleic acid is present in the sample.

In certain embodiments, prior to use of the detection system, thechannel may be free of the sensor compounds (e.g., one or more nucleicacid probes). That is, a manufacturer of the detection system may notpre-treat or modify the channel to include the sensor compound. In thiscase, during use, a user may introduce one or more sensor compounds, forexample in an electrolyte buffer, into the channel and detect areference electrical property value of the channel with the sensorcompound but in the absence of a sample.

In certain other embodiments, prior to use of the detection system, thechannel may be pre-treated or modified so that at least a portion of aninner surface of the channel includes or is coated with a sensorcompound (e.g., one or more nucleic acid capture probes). In oneexample, the manufacturer may detect a reference electrical propertyvalue of the channel modified with the sensor compound and, during use,a user may use the stored reference electrical property value. That is,a manufacturer of the detection system may pre-treat or modify thechannel to include a sensor compound. In this case, a user may need tointroduce the sample and one or more additional sensor compounds intothe channel.

In one example, the user may introduce one or more sensor compounds(e.g., one or more nucleic acid probes) and the sample into the channelconcurrently, for example, in a mixture of the sensor compound and thesample. In this case, a reference electrical property value may bedetected in the channel prior to introduction of the mixture, and anelectrical property value may be detected after introduction of themixture. Comparison of the electrical property value to the referenceelectrical property value may be used to determine if the nucleic acidis present in the sample.

In another example, the user may introduce one or more sensor compounds(e.g., one or more nucleic acid probes) and the sample into the channelconcurrently, for example, in a mixture of the sensor compound and thesample. A stored reference electrical property value characterizing thechannel prior to introduction of the mixture may be retrieved oraccessed from a non-transitory storage medium. An electrical propertyvalue may be detected after introduction of the mixture into thechannel. Comparison of the electrical property value to the storedreference electrical property value may be used to determine if thenucleic acid is present in the sample.

In another example, the user may first introduce one or more sensorcompounds (e.g., one or more nucleic acid probes) into the channel, anddetect a reference electrical property value prior to introduction ofthe sample into the channel. The user may subsequently introduce thesample and optionally, one or more additional sensor compounds, into thechannel, and detect an electrical property value after waiting for atime period after introduction of the sample into the channel.Comparison of the electrical property value to the reference electricalproperty value may be used to determine if the nucleic acid is presentin the sample.

In another example, the user may first introduce one or more sensorcompounds (e.g., one or more nucleic acid probes) into the channel, andmay subsequently introduce the sample and optionally, one or moreadditional sensor compounds, into the channel. The user may then detectan electrical property value after waiting for a time period afterintroduction of the sample into the channel. A stored referenceelectrical property value characterizing the channel prior tointroduction of the sample and all of the sensor compounds may beretrieved or accessed from a non-transitory storage medium. Comparisonof the stored electrical property value to the reference electricalproperty value may be used to determine if the nucleic acid is presentin the sample.

In another example, the user may first introduce the sample into thechannel, and detect a reference electrical property value with only thesample in the channel. The user may subsequently introduce the sensorcompounds (e.g., one or more nucleic acid probes) into the channel, anddetect an electrical property value after waiting for a time periodafter introduction of the sensor compounds into the channel. Comparisonof the electrical property value to the reference electrical propertyvalue may be used to determine if the nucleic acid is present in thesample.

In another example, the user may first introduce the sample into thechannel, and may subsequently introduce the sensor compounds (e.g., oneor more nucleic acid probes) into the channel. The user may then detectan electrical property value after waiting for a time period afterintroduction of the sensor compounds into the channel. A storedreference electrical property value characterizing the channel prior tointroduction of the sample and all of the sensor compounds may beretrieved or accessed from a non-transitory storage medium. Comparisonof the stored electrical property value to the reference electricalproperty value may be used to determine if the nucleic acid is presentin the sample.

In certain other embodiments, prior to use of the detection system, thechannel may be pre-treated or modified so that at least a portion of aninner surface of the channel includes or is coated with a first sensorcompound (e.g., one or more nucleic acid capture probes). That is, amanufacturer of the detection system may pre-treat or modify the channelto include the sensor compound. The manufacturer may detect a referenceelectrical property value of the channel with the first sensor compoundand may store the reference electrical property value on anon-transitory storage medium. During use, the user may introduce thesample and one or more additional sensor compounds (e.g., one or morenucleic acid probes) into the channel and detect an electrical propertyvalue after waiting for a time period after introduction of the sampleinto the channel. The stored reference electrical property value may beaccessed or retrieved from the storage medium. Comparison of theelectrical property value to the reference electrical property value maybe used to determine if the nucleic acid is present in the sample.

In another example, the user may detect a reference electrical propertyvalue of the channel with prior to introducing the sample into thechannel. The user may subsequently introduce the sample into the channeland detect an electrical property value after waiting for a time periodafter introduction of the sample into the channel. Comparison of theelectrical property value to the reference electrical property value maybe used to determine if the nucleic acid is present in the sample.

FIGS. 7A and 7B are flowcharts of an exemplary method 700 for detectinga nucleic acid or nucleotide in a sample.

In step 702, at least a portion of an inner surface of a channel may bepre-treated or covalently modified so that it includes or is coated witha material that enables attachment of a nucleic acid probe. Exemplarymaterials may include, but are not limited to, a silane compound (e.g.,tricholorsilane, triethoxysilane, alkylsilane, perfluoro silane),zwitterionic sultone, poly(6-9)ethylene glycol (Peg), perfluorooctyl,fluorescein, an aldehyde, a graphene compound, and the like. Thecovalent modification of the inner surface of the channel may preventnon-specific absorption of certain molecules. In one example, covalentmodification of the inner surface may enable covalent bonding of one ormore nucleic acid capture probes to the inner surface while preventingnon-specific absorption of other molecules to the inner surface.

The channel modification material may be a silane compound in oneexample. The silane modification may be useful in attaching one or moreprobes, e.g., nucleic acid probes, to the inner surface of the channel.In one exemplary technique of “silanizing” the inner surface, a solutionis produced. The solution may be between 0.1% and 4% v/v (if silane isliquid) or w/v (if silane is a solid) of appropriate chloro-,trichloro-, trimethoxy- or triethoxysilane in the appropriate solvent(e.g. toluene for trimethoxy- or triethoxysilanes, ethanol for chloro-or trichlorosilanes or water with a pH between 3.5 to 5.5 fortrimethoxysilanes). In one example, about 1 mL of triethoxyeldehydesilane may be dissolved in about 24 mL toluene, and the solution may befiltered through a 0.2 micron surfactant free cellulose acetate (SFCA)filter. About 10 μL of the filtered silane solution may be added to aport of the channel and allowed to capillary fill the channel for about5 minutes. This may or may not be observed by light microscopy and maytake between five and forty minutes depending upon the solventcomposition. After capillary filling is complete, about 10 μL of thefiltered silane solution may be added to the remaining ports of thechannel. The entire channel is immersed in the filtered silane solutionand allowed to react for the desired amount of time (for example, about1 to 24 hours) at the desired temperature (for example, about 20° C. to80° C. depending upon the specific silane and solvent composition usedfor the modification). In one example, the channel may be immersed inthe filtered silane solution and heated at about 45° C. for about 18hours. After the desired reaction time is over, the silanization processmay be quenched using one of the following techniques. A catalyticamount of acetic acid may be added to toluene or ethanol-based surfacemodifications in some cases.

In one exemplary technique of quenching, the entire channel may betransferred to a container filled with about 25 mL of 0.2 micron SFCAfiltered ethanol, and stored until the desired time for use or furthermodification. In another exemplary technique of quenching, the channelmay be electrokinetically washed with an appropriate solventcomposition. In one electrokinetic washing technique for toluenemodification of a channel, toluene is electrokinetically driven throughthe channel at a 10V to 1000V differential between electrodes for about5 to 15 minutes, followed by electrokinetically driving ethanol throughthe channel at a 10V to 1000V differential between electrodes for about5 to 15 minutes, followed by electrokinetically driving a 1:1 mixture ofethanol:water through the channel at 10V to 1000V differential betweenelectrodes for about 5 to 15 minutes, followed by a final electrokineticdriving of water through the channel at 10V to 1000V for about 5 to 15minutes. Proper operation of the channel may be confirmed by measuringthe current at 1000V applied field to an added 50 mM sodium boratebuffer in the channel (giving a current reading of approximately 330 nA)and re-addition of ultrapure (e.g., MilliQ ultrapure) water at the sameapplied field affording a current of less than about 20 nA.

In step 704, one or more nucleic acid probes (e.g., a capture probe) maybe attached to at least a portion of the modified inner surface of thechannel. In one embodiment, the nucleic acid probe may be covalentlyattached to the modified inner surface of the channel.

In one example of step 704, the channel modified as in step 702 may beplaced on a hot plate at a low setting for about 15 minutes to removeall ethanol from the channel. About 2 μL of about 1 mM stock 5′hydrazide modified DNA may be mixed with about 198 μL of about pH 4.5buffer containing about 50 mM sodium acetate and 1 mM5-methoxyanthranilic acid. The final DNA concentration in the solutionmay be about 10 μM. About 20 μL of this solution may be added to a portof the modified channel and allowed to capillary fill the channel forabout 40 minutes. Subsequently, about 10 μL of the solution may be addedto the remaining ports of the channel. Loading of the solution in thechannel may be ensured electrokinetically by connecting electrodes tothe ports of the channel and maintaining about a 700 V potentialdifference using a Kiethley 2410 device for about 5 minutes or until astable current is detected. In one example, a stable current of about230 nA may be detected. The solution may be allowed to remain in thechannel to modify the channel for about 3 hours. Subsequently, thechannel may be electrokinetically washed with ultrapure (e.g., MilliQultrapure) water at a 1000 V potential difference between two portsuntil a current of less than about 10 nA is detected. The modifiedchannel may then be stored in a vacuum dessicator until use in the latersteps.

In step 706, a pre-mixture of a sample and a nucleic acid probe (e.g., across-linking target probe) may be prepared. In one example, thecross-linking target probe is selected so that it binds both with thecapture probe provided at the inner surface of the channel in step 704and with the analyte nucleic acid if it is present in the sample. Instep 708, the pre-mixture may be introduced into the channel. In oneexample, the sample may be a human liver total RNA extract (which may ormay not include the analyte GAPD RNA). In this case, the pre-mixture mayinclude a solution containing about 45.5 μL nuclease-free water, about33.3 μL lysis buffer, about 1 μL blocking reagent, about 0.3 μL of anucleic acid probe (e.g., a cross-linking target probe), and about 20 μLof 20 ng/mL human liver total RNA extract that is vortex mixed. About 10μL of this solution may be introduced into the channel through one portand allowed to capillary fill the channel. About 10 μL of the samesolution may then be introduced into another port of the channel.

In step 710, a potential difference may be applied across at least aportion of the length of the channel using a voltage source. In step712, while the potential difference is being applied, one or moreelectrical property values (e.g., current, conductivity, resistivity)may be detected along at least a portion of the length of the channel.In one example, a potential difference of about ±1000 V may be applied,and an electrical current value of about 0.4 μA may be detected.

In order to obtain an accurate and reliable measure of the electricalcurrent, in step 714, an equilibration circuit may be used to analyze afirst set of two or more values of the values that were detected in step712. The equilibration circuit may determine if the values have reachedequilibrium, i.e., have stopped temporally varying outside of apredetermined variance or tolerance range. If it is determined that thevalues have not reached equilibrium, then the method may return to step712 to detect additional values. On the other hand, if it is determinedthat the values have reached equilibrium, then the method may proceed tostep 716. In step 716, the equilibration circuit may select a first orreference value from the first set of values. The first or referencevalue may be used to represent one or more electrical properties of thechannel prior to formation of any aggregate particles in the channel.

In certain other examples, the first value may be measured when thechannel is filled only with a wash buffer and/or only with a diluentbuffer containing no nucleic acids. In one example, at a potentialdifference at ±1000V, the first electrical property value may be acurrent of about 13-19 nA (for a wash buffer) and about 380-400 nA (fora diluent buffer).

In step 718, in some embodiments, the channel may be incubated andwashed with a suitable wash buffer to remove nucleic acids that are notspecifically bound into an aggregate in the channel. Optionally, anelectrical property value may be detected subsequently. In step 720, oneor more additional nucleic acid probes may be introduced into thechannel. Exemplary nucleic acid probes may include one or more labelextenders selected so that they bind directly or indirectly with theanalyte nucleic acid, and/or one or more amplification probes selectedso that they bind with the label extenders. The interactions result inthe formation of an aggregate, which may be electrically conductive insome cases. The electrically conductive aggregate may enhance theelectrical conductivity in the channel and may result in a measurableincrease in an electrical property value (e.g., current, conductivity)and a measurable decrease in another electrical property value (e.g.,resistivity) if the analyte nucleic acid is present in the sample.

In some cases in which multiple nucleic acid probes are sequentiallyintroduced, steps 718 and 720 may be repeated for the introduction ofeach nucleic acid probe.

In step 722, in some embodiments, the channel may be incubated andwashed with a suitable wash buffer to remove nucleic acids that are notspecifically bound into an aggregate formation in the channel. In oneexample, the channel may be sealed and incubated at about 50° C. forabout 90 minutes, and then allowed to cool to room temperature for about45 minutes.

The channel may then be cleaned with a wash buffer until a stablecurrent is detected.

In step 724, a potential difference may be applied across at least aportion of the length of the channel using a voltage source. In step726, while the potential difference is being applied, one or moreelectrical property values along at least a portion of the length of thechannel may be detected.

In order to obtain an accurate and reliable measure of the electricalcurrent, in step 728, an equilibration circuit may be used to analyze asecond set of two or more values that were detected in step 726. Theequilibration module may determine if the values have reachedequilibrium, i.e., have stopped temporally varying outside of apredetermined variance or tolerance range. If it is determined that thevalues have not reached equilibrium, then the method may return to step726 to detect additional values. On the other hand, if it is determinedthat the values have reached equilibrium, the method may proceed to step730.

In step 730, the equilibration circuit may select a second value fromthe second set of values. The second value may be used to represent oneor more electrical properties of the channel after any interactionbetween the nucleic acid and all of the nucleic acid probes. In oneexample, at a potential difference of about ±10 V, a current of about 1μA to about 3.5 μA may be detected if the sample contains the nucleicacid. At a potential difference of about ±100 V, a current of about 3 μAto about 20 μA may be detected if the sample contains the nucleic acid.

In step 732, the comparison circuit may be used to determine adifference between the first or reference value (determined in step 716)and the second value (determined in step 730). In step 734, thecomparison circuit may determine if the difference determined in step732 satisfies a predetermined threshold, for example, if the differenceis above a predetermined value, below a predetermined value, or if thedifference is within a predetermined range. In one example in which theaggregate is electrically conductive, the second electrical propertyvalue may be about 1 μA to about 20 μA greater than the first orreference value, a range of values that indicates formation of anaggregate in the channel that is electrically conductive and thatenhances the electrical conductivity of the channel, thereby indicatingthat the sample included the nucleic acid. In another example, thesecond electrical property value may be about 1 μA to about 20 μA lowerthan the first or reference value, a range of values that indicatesformation of an aggregate in the channel, thereby indicating that thesample included the nucleic acid.

If it is determined in step 734 that the difference between the firstand second values satisfies the predetermined threshold, then thenucleic acid detection circuit may determine in step 740 that the samplecontains the nucleic acid. Subsequently, in step 742, the nucleic aciddetection circuit may store, on a non-transitory computer-readablemedium, an indication that the sample contains the nucleic acid.Alternatively or additionally, in step 742, the nucleic acid detectioncircuit may display, on a display device, an indication that the samplecontains the nucleic acid.

On the other hand, if it is determined in step 734 that the differencebetween the first and second values does not satisfy the predeterminedthreshold, then the nucleic acid detection circuit may determine in step736 that the sample does not contain the nucleic acid. Subsequently, instep 738, the nucleic acid detection circuit may store, on anon-transitory computer-readable medium, an indication that the sampledoes not contain the nucleic acid. Alternatively or additionally, instep 738, the nucleic acid detection circuit may display, on a displaydevice, an indication that the sample does not contain the nucleic acid.

In one example of steps 718-732, the channel may be sealed and incubatedin an oven at about 55° C. for about 16 hours and then removed from theoven. About 10 μL of a wash buffer may be electrokinetically driventhrough the channel for about 10 minutes, a potential difference ofabout ±100 V may be applied, and an electrical property value may bedetected. An exemplary electrical property value detected may be currentranging from about 6 μA to about 7.5 μA. Subsequently, about 10 μL of asolution containing 1 μL of a nucleic acid probe (e.g., apre-amplification probe) in about 1 mL of diluent buffer may beelectrokinetically driven into the channel. A potential difference ofabout ±100 V may be applied, and an electrical property value may bedetected. An exemplary electrical property value detected may be currentranging from about 5.8 μA to about 7.5 μA.

The channel may then be sealed and incubated at about 55° C. for aboutan hour. About 10 μL of a wash buffer may be electrokinetically driventhrough the channel for about 10 minutes, a potential difference ofabout ±100 V may be applied, and an electrical property value may bedetected. An exemplary electrical property value detected may be currentranging from about 2.8 μA to about 3.2 μA. Subsequently, about 10 μL ofa solution containing 1 μL of a nucleic acid probe (e.g., anamplification probe) in about 1 mL of diluent buffer may beelectrokinetically driven into the channel until the current is detectedto be stable. A potential difference of about ±100 V may be applied, andan electrical property value may be detected. An exemplary electricalproperty value detected may be current of about 4 μA.

The channel may then be sealed and incubated at about 55° C. for aboutan hour. About 10 μL of a wash buffer may be electrokinetically driventhrough the channel for about 10 minutes, a potential difference ofabout ±100 V may be applied, and an electrical property value may bedetected. An exemplary electrical property value detected may be currentranging from about 5 μA to about 20 μA. Subsequently, about 10 μL of asolution containing 1 μL of a nucleic acid probe (e.g., a labelextender) in about 1 mL of diluent buffer may be electrokineticallydriven into the channel until the current is detected to be stable. Apotential difference of about ±100 V may be applied, and an electricalproperty value may be detected. An exemplary electrical property valuedetected may be current ranging from about 3 μA to about 10 μA.

In certain embodiments, the channel may be reused for subsequent testingof samples. In one exemplary embodiment, in step 746, a de-aggregationagent (e.g., a nucleic acid surface cleavage or degradation buffer) maybe introduced into the channel to cause the aggregate to disintegrate sothat the channel may be reused. In step 748, the channel may be filledwith an electrolyte buffer to flush out the aggregate from the channeland one or more electrical properties (e.g., current) may be detected toensure that the aggregate has been cleared from the channel. In oneexample, a marked reduction in the electrical current may indicate thatan electrically conductive aggregate has been cleared from the channel.

In one example of steps 746 and 748, the channel with the aggregate iselectrokinetically loaded with a buffer containing 50 mM sodiumphosphates (pH 7.4), 1 mM 5-methoxyanthranilic acid and 5 mMhydroxylamine hydrochloride until a stable current is obtained(+/−100V=1.4−1.7 μA). The entire channel is then allowed to incubate inthis buffer for about 18 hours at room temperature, after which thecurrent is again measured until stable (+1000V=86−87 nA, −1000V=63−64nA). The significant decrease in current (from about 1.4-1.7 μA beforeintroduction of the surface cleavage buffer to about 63-90 nA afterwashing with the surface cleavage buffer) is indicative of clearing ofthe electrically conductive aggregate.

In certain embodiments, in step 744, prior to disintegration of theaggregate, an absolute or relative concentration of a nucleic acid maybe determined based on an electrical property value of the channel. Theconcentration of the nucleic acid may be determined in such a mannerbecause the channels of exemplary detection systems have a high innersurface area to volume ratio. At low concentrations of the nucleic acid,electrical conductivity in the channel is dominated by surface charges.As such, measurements of electrical properties of the channel can enabledistinction between different ions. As a result, unique and sensitivemeasurements of the bulk flow in the channel can be used to determineinformation on the surface charges at the inner surface of the channel.Exemplary embodiments may thus compute predetermined ranges ofelectrical property values of the channel that are characteristic of thenucleic acid particles given the dimensions of the channel and atdifferent concentrations of the nucleic acid. These predetermined valuesmay then be used to determine an unknown concentration of the nucleicacid in a sample.

Detailed information on surface charges in the channel for differentions is presented in the following papers, the entire contents of whichare expressly incorporated herein by reference: “Surface-dependentchemical equilibrium constants and capacitances for bare and3-cyanopropyldimethylchlorosilane coated silica nanochannels,” M. B.Andersen, J. Frey, S. Pennathur and H. Bruus, J., Colloid Interface Sci.353, 301-310 (2011), and “Hydronium-domination ion transport incarbon-dioxide-saturated electrolytes at low salt concentrations innanochannels,” K. L. Jensen, J. T. Kristensen, A. M. Crumrine, M. B.Andersen, H. Bruus and S. Pennathur, Phys. Review E. 83, 5, 056307.

FIG. 5 is a schematic drawing of the inside of a channel including aninner surface of the channel 502, an immobile layer of fluid 504 lyingimmediately adjacent to the inner surface of the channel, a diffusivelayer of fluid 506 lying immediately adjacent to the immobile layer, anda bulk fluid flow layer 508 lying immediately adjacent to the diffusivelayer. Exemplary ions are represented in each of the fluid layers. Uponapplication of a potential difference across the length of the channel,an electrical property value may be detected along at least a portion ofthe channel (for example, by the nucleic acid detection circuit 122).The comparison circuit 124 may be used to compare the measuredelectrical property value to a predetermined range of electricalproperty values that correspond to a particular concentration or rangeof concentration values of the nucleic acid. The concentrationdetermined may be an absolute concentration of the nucleic acid or arelative concentration of the nucleic acid with respect to theconcentrations of one or more other substances in the channel.

FIGS. 6A and 6B are graphs showing conductivity values measured in achannel for different test cases. In each test case, a differentrelative concentration of an analyte relative to concentrations of twoadditional substances (in this case, ammonium and hydrogen peroxide) isused, and the corresponding conductivity value is determined in thechannel. In one embodiment, Standard Clean 1 or SC1 is used a solutionin the test cases. Details of SC1 can be found athttp://en.wikipedia.org/wiki/RCA_clean, the entire contents of which areexpressly incorporated herein by reference. The ratios of concentrationsamong the three substances in the test cases represented in FIGS. 6A and6B are presented in Table 1 above.

The lower the concentration of an analyte, the easier it is to measuredifferences in relative concentrations between the analyte and othersubstances. For example, at concentration ratios of about 1000:1:1,detection sensitivity on the order of 1-10 ppm may be achieved in theexemplary detection system. At concentration ratios of about 350:1:1,detection sensitivity on the order of 100 ppm may be achieved. Atconcentration ratios of about 5:1:1, detection sensitivity on the orderof 10,000 ppm may be achieved.

Table 3 presented below summarizes exemplary electrical current valuesthat may be detected at different stages of the method of FIGS. 7A and7B. One of ordinary skill in the art will recognize that the exemplarynumerical values presented in Table 3 are merely for illustrativepurposes and are not intended to limit the scope of the invention.

TABLE 3 Changes in electrical current during detection of nucleic acidsStep Applied Voltage Measured Current Introduction of sample and capture+1000 V 409-410 nA components (step 708) −1000 V 403-404 nA Wash ofsample and capture +/−100 V 6-7.5 μA components after 16 hr incubationat 55° C. (Step 716) Loading of preamplifier probes +/−100 V 5.8-7.5 μA(Step 720) Washing of preamplifier probes +/−100 V 2.8-3.2 μA after 1 hrincubation at 55° C. (Step 718) Loading of amplifier probes +/−100 V 4μA (Step 720) Washing of amplifier probes after +/−100 V 5-20 μA 1 hrincubation at 55° C. (Step 718) Loading of label probes +100 V 30 μA(Step 720) −100 V 3-10 μA Washing of label probes after +10 V 0.9-1.4 μAincubation (Step 718) −10 V 2-3.5 μA Loading of surface cleavage/ +/−100V 1.4-1.7 μA degradation buffer (Step 746) Washing of surface cleavagebuffer +1000 V 86-87 nA (Step 748) −1000 V 63-64 nA

In one example, one or more electrical properties of a channel with nosurface modification were detected in which only buffers with no addednucleic acids were exposed to the channel. Table 4 summarizes the stablecurrents that were detected when a wash buffer and a diluent buffer werepresent in the channel.

TABLE 4 Control buffer current measurements in channel Buffer AppliedVoltage Measured Current Wash buffer +1000 V  19 nA −1000 V  13 nADiluent buffer +1000 V 396 nA −1000 V 385 nA

FIG. 8 is a flowchart illustrating a general exemplary method 800 fordetecting the presence or absence of a nucleic acid in a sample. In step802, a sample may be introduced into a channel of a detection system,the channel having a length and a width, the length substantiallygreater than the width. In step 804, an electrical property value of anelectrical property (e.g., current, conductivity, resistance) may bemeasured along at least a portion of the length of the channel after thesample is introduced into the channel. In step 806, a referenceelectrical property value may be accessed. The reference electricalproperty value may be associated with the electrical property detectedin step 804 along at least a portion of the length of the channel priorto introduction of the sample into the channel. In step 808, theelectrical property value measured in step 804 may be compared to thereference electrical property value accessed in step 806. In step 810,based on the comparison in step 808, presence or absence of the nucleicacid in the sample may be determined.

FIG. 9 is a flowchart illustrating a general exemplary method 900 fordetecting the presence or absence of a nucleic acid in a sample. In step902, one or more electrical property values of one or more electricalproperties (e.g., current, conductivity, resistance) may be measuredalong at least a portion of the length of a channel, the channel havinga length and a width, the length substantially greater than the width.In step 904, a reference channel electrical property value may bedetermined based on the electrical property values of the channelmeasured in step 902. In step 906, a sample may be introduced into thechannel. In step 908, one or more electrical property values of one ormore electrical properties (e.g., current, conductivity, resistance) maybe measured along at least a portion of the length of the channel afterintroduction of the sample into the channel. In step 910, a samplechannel electrical property value may be determined based on the one ormore electrical property values measured in step 908. In step 912, thesample channel electrical property value determined in step 910 may becompared to the reference channel electrical property value determinedin step 904. In step 914, based on the comparison in step 912, presenceor absence of the nucleic acid in the sample may be determined.

FIG. 10 is a flowchart illustrating a general exemplary method 1000 fordetecting the presence or absence of a nucleic acid in a sample. In step1002, a mixture of a sample and one or more sensor compounds may beintroduced into a channel, the channel having a length and a width, thelength substantially greater than the width. In step 1004, an electricalproperty value of an electrical property (e.g., current, conductivity,resistance) may be measured along at least a portion of the length ofthe channel after the sample and all of the sensor compounds areintroduced into the channel. In step 1006, a reference electricalproperty value may be accessed. The reference electrical property valuemay be associated with the electrical property detected in step 1004along at least a portion of the length of the channel prior tointroduction of the sample and all of the sensor compounds into thechannel. In step 1008, any differences between the electrical propertyvalue measured in step 1004 and the reference electrical property valueaccessed in step 1006 may be determined. In step 1010, based on thedifferences, if any, determined in step 1008, presence or absence of thenucleic acid in the sample may be determined.

FIG. 11 is a flowchart illustrating a general exemplary method 1100 fordetecting the presence or absence of a nucleic acid in a sample. In step1102, one or more sensor compounds may be introduced into a channel, thechannel having a length and a width, the length substantially greaterthan the width. In step 1104, one or more electrical properties (e.g.,current, conductivity, resistance) may be measured along at least aportion of the length of a channel. In step 1106, a reference channelelectrical property value may be determined based on the electricalproperties of the channel measured in step 1104. In step 1108, a samplemay be introduced into the channel. In step 1110, one or more electricalproperties (e.g., current, conductivity, resistance) may be measuredalong at least a portion of the length of a channel. In step 1112, anelectrical property value of the channel may be determined based on theone or more electrical properties measured in step 1110. In step 1114,any differences between the electrical property value determined in step1112 and the reference channel electrical property value determined instep 1106 may be determined. In step 1116, based on the differences, ifany, determined in step 1114, presence or absence of the nucleic acid inthe sample may be determined.

FIG. 12 is a flowchart illustrating a general exemplary method 1200 fordetecting the presence or absence of a nucleic acid in a sample. In step1202, one or more sensor compounds may be introduced into a channel, thechannel having a length and a width, the length substantially greaterthan the width. In step 1204, a sample may be introduced into thechannel. In step 1206, one or more electrical properties (e.g., current,conductivity, resistance) may be measured along at least a portion ofthe length of a channel. In step 1208, an electrical property value ofthe channel may be determined based on the one or more electricalproperties measured in step 1206. In step 1210, a reference channelelectrical property value may be accessed. The reference channelelectrical property value may be measured prior to introduction of allof the sensor compounds and the sample into the channel. In step 1212,any differences between the electrical property value determined in step1208 and the reference channel electrical property value accessed instep 1210 may be determined. In step 1214, based on the differences, ifany, determined in step 1212, presence or absence of the nucleic acid inthe sample may be determined.

FIG. 13 is a flowchart illustrating a general exemplary method 1300 fordetecting the presence or absence of a nucleic acid in a sample. In step1302, a sample may be introduced into a channel of a detection system,the channel having a length and a width, the length substantiallygreater than the width. In step 1304, one or more electrical properties(e.g., current, conductivity, resistance) may be measured along at leasta portion of the length of the channel after the sample is introducedinto the channel. In step 1306, a reference channel electrical propertyvalue may be determined based on the one or more electrical propertiesmeasured in step 1304. In step 1308, one or more sensor compounds may beintroduced into the channel. In step 1310, one or more electricalproperties (e.g., current, conductivity, resistance) may be measuredalong at least a portion of the length of the channel after the sensorcompounds are introduced into the channel. In step 1312, an electricalproperty value may be determined based on the one or more electricalproperties measured in step 1310 after all of the sensor compounds andthe sample are introduced into the channel. In step 1314, anydifferences between the electrical property value determined in step1312 and the reference channel electrical property value determined instep 1306 may be determined. In step 1316, based on the differences, ifany, determined in step 1314, presence or absence of the nucleic acid inthe sample may be determined.

FIG. 14 is a flowchart illustrating a general exemplary method 1400 fordetecting the presence or absence of a nucleic acid in a sample. In step1402, a sample may be introduced into a channel of a detection system,the channel having a length and a width, the length substantiallygreater than the width. In step 1404, one or more sensor compounds maybe introduced into the channel. In step 1406, one or more electricalproperties (e.g., current, conductivity, resistance) may be measuredalong at least a portion of the length of the channel after the sampleand all of the sensor compounds are introduced into the channel. In step1408, an electrical property value may be determined based on the one ormore electrical properties measured in step 1406 after all of the sensorcompounds and the sample are introduced into the channel. In step 1410,a reference channel electrical property value may be accessed. Thereference channel electrical property value may be measured prior tointroduction of all of the sensor compounds and the sample into thechannel. In step 1412, any differences between the electrical propertyvalue determined in step 1408 and the reference channel electricalproperty value accessed in step 1410 may be determined. In step 1414,based on the differences, if any, determined in step 1412, presence orabsence of the nucleic acid in the sample may be determined.

FIG. 15 is a flowchart illustrating a general exemplary method 1500 fordetecting the presence or absence of a nucleic acid in a sample. In step1502, at least a portion of an inner surface of a channel may bemodified or treated with a material that may facilitate or enablespecific covalent attachment of one or more nucleic acid probes to theinner surface of the channel. The channel may have a length and a width,the length substantially greater than the width. Exemplary materialsthat may be used to modify the inner surface of the channel include, butare not limited to, a silane compound (e.g., tricholorsilane,alkylsilane, triethoxysilane, perfluoro silane), zwitterionic sultone,poly(6-9)ethylene glycol (Peg), perfluorooctyl, fluorescein, analdehyde, a graphene compound, and the like. The covalent modificationof the inner surface of the channel may prevent non-specific absorptionof certain molecules, for example, molecules other than nucleic acidprobes. In step 1504, at least a portion of the inner surface of thechannel may be coated or provided with one or more nucleic acid probes.The nucleic acid probes may be covalently attached to the modifiedportion of the inner surface. In step 1506, one or more electricalproperties (e.g., current, conductivity, resistance) may be measuredalong at least a portion of the length of a channel. In step 1508, areference channel electrical property value may be determined based onthe one or more electrical properties measured in step 1506. In step1510, the reference channel electrical property value may be stored on anon-transitory storage medium for use in determining whether a nucleicacid is present or absent in the sample.

FIG. 16 is a schematic of exemplary nucleic acid probes that may be usedin the methods of FIGS. 7A, 7B, 8-15, 17A and 17B. FIG. 16 illustratesan inner surface 1602 of a channel 1604 which is pre-treated or modified(for example, with molecules of a silane compound) to enable attachmentof one or more nucleic acid probes (e.g., capture probes 1606) to theinner surface 1602. The capture probes 1606 are selected so that theybind with one or more cross-linking target probes 1608, and the targetprobes 1608 are selected so that they bind both with a particularnucleic acid 1610 (which is the analyte being tested for, and which maybe a viral DNA in one example) and the capture probes 1606.

A sample (which may or may not contain the nucleic acid 1610) and thetarget probes 1608 may be introduced into the channel concurrently orsequentially. Interactions among the nucleic acid 1610, the targetprobes 1608 and the capture probes 1606 may result in an aggregate 1612in the channel. In certain embodiments, one or more additional nucleicacid probes (e.g., one or more label extenders 1614) may be introducedinto the channel. The label extenders 1614 are selected so that theybind with the nucleic acid 1610 in the aggregate 1612 to form a morecomplex aggregate 1616. One or more additional nucleic acid probes(e.g., one or more amplification probes 1618) may also be introducedinto the channel. The amplification probes 1618 are selected so thatthey bind with the label extenders 1614 in the aggregate 1616 to form amore complex aggregate 1620 that may be electrically conductive in somecases. The electrically conductive aggregate 1620 may enhance theelectrical pathway along at least a portion of the length of thechannel, and may result in a measurable increase in an electricalproperty value (e.g., current, conductivity) and a measurable decreasein another electrical property value (e.g., resistivity) compared to areference value, if the nucleic acid is present in the sample. Thus,detection of an increased electrical current or conductivity in thechannel, relative to a reference value, may indicate the presence of thenucleic acid 1610 in a sample. Similarly, detection of a decreasedresistivity relative to a reference value may indicate the presence ofthe nucleic acid 1610 in a sample.

Another exemplary technique for detecting a nucleic acid may involvedetection of the presence of a diode-like behavior in the channel thatis caused by the formation of a nucleic acid aggregate in the channel.In the absence of an aggregate, application of a potential differencehaving a substantially similar magnitude (e.g., +500 V) may result in asubstantially same magnitude of an electrical property (e.g., current)detected along the length of the channel regardless of the direction ofapplication of the potential difference or electric field. If thepotential difference is applied across the length of the channel in afirst direction along the length of the channel (e.g., such that thepositive electrode is at an input port 110 at or near a first end of thechannel and such that the negative electrode is at an output port 112 ator near a second end of the channel), the resulting current may besubstantially equal in magnitude to the resultant current if thepotential difference is applied in the opposite direction (e.g., suchthat the positive electrode is at the output port 112 and such that thenegative electrode is at the input port 110).

Formation of an aggregate in the channel may cause a diode-like behaviorin which reversal of the direction of the applied potential differenceor electric field causes a change in the electrical property detected inthe channel. The diode-like behavior causes the detected electricalcurrent to vary in magnitude with the direction of the electric field.When the electric field or potential difference is applied in the firstdirection, the magnitude of the electrical current may be different inmagnitude than when the potential different or electric field is appliedin the opposite direction. Thus, comparison between a first electricalproperty value (detected when a potential difference is applied in afirst direction along the channel length) and a second electricalproperty value (detected when a potential difference is applied in asecond opposite direction along the channel length) may enable detectionof an aggregate, and thereby detection of a nucleic acid in the sample.If the first and second electrical property values are substantiallyequal in magnitude, then it may be determined that the sample does notcontain the nucleic acid. On the other hand, if the first and secondelectrical property values are substantially unequal in magnitude, thenit may be determined that the sample contains the nucleic acid. In otherwords, the sum of the values of the electrical property (positive in onedirection, negative in the other direction) is substantially zero in theabsence of an aggregate and substantially non-zero in the presence of anaggregate.

FIGS. 17A and 17B are flowcharts illustrating an exemplary method 1850for detecting the presence or absence of the nucleic acid in a sample.In step 1852, one or more nucleic acid probes and a sample may beintroduced into the channel using any suitable technique, for example,capillary filing or electro-kinetic filling. The nucleic acid probes andthe sample may be introduced concurrently or separately. In oneembodiment, at least a portion of an inner surface of the channel may betreated to include or be coated with a nucleic acid probe (e.g., acapture probe).

In step 1854, a potential difference may be applied across at least aportion of the length of the channel using a voltage source in a firstdirection along the channel length (y-axis). In step 1856, while thepotential difference is being applied, one or more electrical propertiesvalues (e.g., the electrical current and/or conductivity) along at leasta portion of the length of the channel may be detected. In some cases,the electrical current and/or conductivity may be directly measured. Inother cases, a measure indicating the electrical current and/or ameasure indicating the electrical conductivity may be detected.

In order to obtain an accurate and reliable measure of the electricalproperties, in step 1858, a first set of two or more values that weredetected in step 1856 may be continually or periodically monitored. Itmay be determined if the electrical property values have reachedequilibrium, i.e., has stopped varying outside of a predeterminedvariance or tolerance range. If it is determined that the electricalproperty values have not reached equilibrium, then the method may returnto step 1856 to detect additional electrical property values. On theother hand, if it is determined that the electrical property values havereached equilibrium, then the method may proceed to step 1860.

In step 1860, a first value may be selected from the first set ofelectrical property. The first electrical property value may be used torepresent the one or more electrical properties (e.g., electricalcurrent or conductivity) of the channel when an electric field isapplied in a first direction along the channel length (y-axis).

In step 1862, a potential difference may be applied across at least aportion of the length of the channel using a voltage source in a secondopposite direction along the channel length (y-axis). The seconddirection may be substantially opposite to the first direction. In step1864, while the potential difference is being applied, one or moreelectrical properties (e.g., electrical current and/or conductivity)along at least a portion of the length of the channel may be detected.In some cases, the electrical current and/or conductivity may bedirectly measured. In other cases, a measure indicating the electricalcurrent and/or a measure indicating the electrical conductivity may bedetected.

In order to obtain an accurate and reliable measure of the electricalproperties, in step 1866, a second set of two or more values that weredetected in step 1864 may be continually or periodically monitored. Itmay be determined if the electrical property values have reachedequilibrium, i.e., has stopped temporally varying outside of apredetermined variance or tolerance range. If it is determined that theelectrical property values have not reached equilibrium, then the methodmay return to step 1864 to detect additional values. On the other hand,if it is determined that the electrical property values have reachedequilibrium, then the method may proceed to step 1868. In step 1868, asecond value may be selected from the second set of values of theelectrical property. The second value may be used to represent the oneor more electrical properties (e.g., electrical current or conductivity)along at least a portion of the length of the channel after both thesample and the sensor compound have been introduced into the channel.

In step 1870, a difference between the magnitude of the first value(determined in step 1860) and the magnitude of the second value(determined in step 1868) may be determined. In step 1872, it may bedetermined if the difference determined in step 1870 satisfies apredetermined threshold, for example, if the difference is above apredetermined value, below a predetermined value, or if the differenceis within a predetermined range.

If it is determined in step 1872 that the difference between the firstand second values satisfies the predetermined threshold (e.g., that thedifference in magnitudes is substantially non-zero), then it may bedetermined in step 1878 that the sample contains the nucleic acid.Subsequently, in step 1880, an indication that the sample contains thenucleic acid may be stored on a non-transitory storage medium.Alternatively or additionally, in step 1880, an indication that thesample contains the nucleic acid may be displayed on a display device.

On the other hand, if it is determined in step 1872 that the differencebetween the first and second values does not satisfy the predeterminedthreshold (e.g., that the difference in magnitudes is substantiallyzero), then it may be determined in step 1874 that the sample does notcontain the nucleic acid. Subsequently, in step 1876, an indication thatthe sample does not contain the nucleic acid may be stored on anon-transitory storage medium. Alternatively or additionally, in step1876, an indication that the sample does not contain the nucleic acidmay be displayed on a display device.

In certain cases, if the difference in magnitude between the first andsecond values is greater than the threshold, then it may be determinedthat the sample contains the nucleic acid. Otherwise, it may bedetermined that the sample does not contain the nucleic acid. In certainnon-limiting examples, the threshold may range from approximately 1 nAto approximately 10 nA.

In certain embodiments, the channel may be prepared for reuse forsubsequent testing of samples. In step 1884, a de-aggregation agent maybe introduced into the channel using any suitable technique, forexample, capillary filing or electro-kinetic filling. The de-aggregationagent may be selected so that interaction between the de-aggregationagent and the aggregate formed in the channel causes the aggregate todissolve or disintegrate. The channel may be filled with an electrolytebuffer to flush out the channel and allow a sample and a sensor compoundto be introduced into the channel.

In certain embodiments, in step 1882, prior to disintegration of theaggregate, an absolute or relative concentration of the nucleic acid maybe determined based on an electrical property value of the channel. Theconcentration of the nucleic acid may be determined in such a mannerbecause the channels of exemplary detection systems have a high innersurface area to volume ratio. At low concentrations of the nucleic acid,electrical conductivity in the channel is dominated by surface charges.As such, measurements of electrical properties of the channel can enabledistinction between different ions. As a result, unique and sensitivemeasurements of the bulk flow in the channel can be used to determineinformation on the surface charges at the inner surface of the channel.Exemplary embodiments may thus compute predetermined ranges ofelectrical property values of the channel that are characteristic of thenucleic acid given the dimensions of the channel and at differentconcentrations of the nucleic acid. These predetermined values may thenbe used to determine an unknown concentration of the nucleic acid in asample.

IV. Exemplary Processors and Computing Devices

Systems and methods disclosed herein may include one or moreprogrammable processors, processing units and computing devices havingassociated therewith executable computer-executable instructions held orencoded on one or more non-transitory computer readable media, RAM, ROM,hard drive, and/or hardware. In exemplary embodiments, the hardware,firmware and/or executable code may be provided, for example, as upgrademodule(s) for use in conjunction with existing infrastructure (forexample, existing devices/processing units). Hardware may, for example,include components and/or logic circuitry for executing the embodimentstaught herein as a computing process.

Displays and/or other feedback means may also be included, for example,for rendering a graphical user interface, according to the presentdisclosure. The displays and/or other feedback means may be stand-aloneequipment or may be included as one or more components/modules of theprocessing unit(s).

The actual computer-executable code or control hardware that may be usedto implement some of the present embodiments is not intended to limitthe scope of such embodiments. For example, certain aspects of theembodiments described herein may be implemented in code using anysuitable programming language type such as, for example, the MATLABtechnical computing language, the LABVIEW graphical programminglanguage, assembly code, C, C# or C++ using, for example, conventionalor object-oriented programming techniques. Such computer-executable codemay be stored or held on any type of suitable non-transitorycomputer-readable medium or media, such as, a magnetic or opticalstorage medium.

As used herein, a “processor,” “processing unit,” “computer” or“computer system” may be, for example, a wireless or wire line varietyof a microcomputer, minicomputer, server, mainframe, laptop, personaldata assistant (PDA), wireless e-mail device (for example, “BlackBerry,”“Android” or “Apple,” trade-designated devices), cellular phone, pager,processor, fax machine, scanner, or any other programmable deviceconfigured to transmit and receive data over a network. Computer systemsdisclosed herein may include memory for storing certain softwareapplications used in obtaining, processing and communicating data. Itcan be appreciated that such memory may be internal or external to thedisclosed embodiments. The memory may also include a non-transitorystorage medium for storing computer-executable instructions or code,including a hard disk, an optical disk, floppy disk, ROM (read onlymemory), RAM (random access memory), PROM (programmable ROM), EEPROM(electrically erasable PROM), flash memory storage devices, or the like.

FIG. 18 depicts a block diagram representing an exemplary computingdevice 1700 that may be used to implement the systems and methodsdisclosed herein. In certain embodiments, the processor 130 illustratedin FIGS. 1A and 1B may be configured as or may implement certainfunctionality and/or components of the computing device 1700. In certainembodiments, the nucleic acid detection circuit 122 may be configured asor may implement certain functionality and/or components of thecomputing device 1700.

The computing device 1700 may be any computer system, such as aworkstation, desktop computer, server, laptop, handheld computer, tabletcomputer (e.g., the iPad™ tablet computer), mobile computing orcommunication device (e.g., the iPhone™ mobile communication device, theAndroid™ mobile communication device, and the like), or other form ofcomputing or telecommunications device that is capable of communicationand that has sufficient processor power and memory capacity to performthe operations described herein. In exemplary embodiments, a distributedcomputational system may include a plurality of such computing devices.

The computing device 1700 may include one or more non-transitorycomputer-readable media having encoded thereon one or morecomputer-executable instructions or software for implementing theexemplary methods described herein. The non-transitory computer-readablemedia may include, but are not limited to, one or more types of hardwarememory and other tangible media (for example, one or more magneticstorage disks, one or more optical disks, one or more USB flash drives),and the like. For example, memory 1706 included in the computing device1700 may store computer-readable and computer-executable instructions orsoftware for implementing functionality of a nucleic acid detectioncircuit 122 as described herein. The computing device 1700 may alsoinclude processor 1702 and associated core 1704, and in someembodiments, one or more additional processor(s) 1702′ and associatedcore(s) 1704′ (for example, in the case of computer systems havingmultiple processors/cores), for executing computer-readable andcomputer-executable instructions or software stored in the memory 1702and other programs for controlling system hardware. Processor 1702 andprocessor(s) 1702′ may each be a single core processor or a multiplecore (1704 and 1704′) processor.

Virtualization may be employed in the computing device 1700 so thatinfrastructure and resources in the computing device may be shareddynamically. A virtual machine 1714 may be provided to handle a processrunning on multiple processors so that the process appears to be usingonly one computing resource rather than multiple computing resources.Multiple virtual machines may also be used with one processor.

Memory 1706 may include a non-transitory computer system memory orrandom access memory, such as DRAM, SRAM, EDO RAM, and the like. Memory1706 may include other types of memory as well, or combinations thereof.

A user may interact with the computing device 1700 through a visualdisplay device 1718, such as a screen or monitor, which may display oneor more graphical user interfaces 1720 provided in accordance withexemplary embodiments described herein. The visual display device 1718may also display other aspects, elements and/or information or dataassociated with exemplary embodiments. In certain cases, the visualdisplay device 1718 may display value of one or more electricalproperties detected in an exemplary nucleic acid detection system ormethod. In certain cases, the visual display device 1718 may display anindication of whether a sample contains or does not contain the nucleicacid. In certain embodiments, other types of interfaces may be providedto communicate the same information, for example, a sound alarm that maybe activated if the nucleic acid is determined to be present in asample.

The computing device 1700 may include other I/O devices for receivinginput from a user, for example, a keyboard or any suitable multi-pointtouch interface 1708 or pointing device 1710 (e.g., a mouse, a user'sfinger interfacing directly with a display device). As used herein, a“pointing device” is any suitable input interface, specifically, a humaninterface device, that allows a user to input spatial data to acomputing system or device. In an exemplary embodiment, the pointingdevice may allow a user to provide input to the computer using physicalgestures, for example, pointing, clicking, dragging, dropping, and thelike. Exemplary pointing devices may include, but are not limited to, amouse, a touchpad, a finger of the user interfacing directly with adisplay device, and the like.

The keyboard 1708 and the pointing device 1710 may be coupled to thevisual display device 1718. The computing device 1700 may include othersuitable conventional I/O peripherals. The I/O devices may facilitateimplementation of the one or more graphical user interfaces 1720, forexample, implement one or more of the graphical user interfacesdescribed herein.

The computing device 1700 may include one or more storage devices 1724,such as a durable disk storage (which may include any suitable opticalor magnetic durable storage device, e.g., RAM, ROM, Flash, USB drive, orother semiconductor-based storage medium), a hard-drive, CD-ROM, orother computer readable media, for storing data and computer-readableinstructions and/or software that implement exemplary embodiments astaught herein. In exemplary embodiments, the one or more storage devices1724 may provide storage for data that may be generated by the systemsand methods of the present disclosure. The one or more storage devices1724 may be provided on the computing device 1700 and/or providedseparately or remotely from the computing device 1700.

The computing device 1700 may include a network interface 1712configured to interface via one or more network devices 1722 with one ormore networks, for example, Local Area Network (LAN), Wide Area Network(WAN) or the Internet through a variety of connections including, butnot limited to, standard telephone lines, LAN or WAN links (for example,802.11, T1, T3, 56 kb, X.25), broadband connections (for example, ISDN,Frame Relay, ATM), wireless connections, controller area network (CAN),or some combination of any or all of the above. The network interface1712 may include a built-in network adapter, network interface card,PCMCIA network card, card bus network adapter, wireless network adapter,USB network adapter, modem or any other device suitable for interfacingthe computing device 1700 to any type of network capable ofcommunication and performing the operations described herein. Thenetwork device 1722 may include one or more suitable devices forreceiving and transmitting communications over the network including,but not limited to, one or more receivers, one or more transmitters, oneor more transceivers, one or more antennae, and the like.

The computing device 1700 may run any operating system 1716, such as anyof the versions of the Microsoft® Windows® operating systems, thedifferent releases of the Unix and Linux operating systems, any versionof the MacOS® for Macintosh computers, any embedded operating system,any real-time operating system, any open source operating system, anyproprietary operating system, any operating systems for mobile computingdevices, or any other operating system capable of running on thecomputing device and performing the operations described herein. Inexemplary embodiments, the operating system 1716 may be run in nativemode or emulated mode. In an exemplary embodiment, the operating system1716 may be run on one or more cloud machine instances.

One of ordinary skill in the art will recognize that exemplary computingdevice 1700 may include more or fewer modules than those shown in FIG.18.

In describing exemplary embodiments, specific terminology is used forthe sake of clarity. For purposes of description, each specific term isintended to, at least, include all technical and functional equivalentsthat operate in a similar manner to accomplish a similar purpose.Additionally, in some instances where a particular exemplary embodimentincludes a plurality of system elements or method steps, those elementsor steps may be replaced with a single element or step. Likewise, asingle element or step may be replaced with a plurality of elements orsteps that serve the same purpose. Further, where parameters for variousproperties are specified herein for exemplary embodiments, thoseparameters may be adjusted up or down by 1/20th, 1/10th, ⅕th, ⅓rd, ½nd,and the like, or by rounded-off approximations thereof, unless otherwisespecified. Moreover, while exemplary embodiments have been shown anddescribed with references to particular embodiments thereof, those ofordinary skill in the art will understand that various substitutions andalterations in form and details may be made therein without departingfrom the scope of the invention. Further still, other aspects, functionsand advantages are also within the scope of the invention.

Exemplary flowcharts are provided herein for illustrative purposes andare non-limiting examples of methods. One of ordinary skill in the artwill recognize that exemplary methods may include more or fewer stepsthan those illustrated in the exemplary flowcharts, and that the stepsin the exemplary flowcharts may be performed in a different order thanshown.

Blocks of the block diagram and the flow chart illustrations supportcombinations of means for performing the specified functions,combinations of steps for performing the specified functions and programinstruction means for performing the specified functions. It will alsobe understood that some or all of the blocks/steps of the circuitdiagram and process flowchart, and combinations of the blocks/steps inthe circuit diagram and process flowcharts, can be implemented byspecial purpose hardware-based computer systems that perform thespecified functions or steps, or combinations of special purposehardware and computer instructions. Exemplary systems may include moreor fewer modules than those illustrated in the exemplary block diagrams.

Many modifications, combinations and other embodiments of the inventionsset forth herein will come to mind to one skilled in the art to whichthese embodiments of the invention pertain having the benefit of theteachings presented in the foregoing descriptions and the associateddrawings. Therefore, it is to be understood that the embodiments of theinvention are not to be limited to the specific embodiments disclosedand that modifications, combinations and other embodiments are intendedto be included within the scope of the appended claims. Althoughspecific terms are employed herein, they are used in a generic anddescriptive sense only and not for purposes of limitation.

What is claimed is:
 1. A method for detecting the presence or absence ofa nucleic acid in a sample, the method comprising: introducing a sampleinto a channel, the channel having a length and a width, the lengthsubstantially greater than the width; measuring an electrical propertyvalue of an electrical property along at least a portion of the lengthof the channel after the sample is introduced into the channel;accessing a reference electrical property value, the referenceelectrical property value associated with the electrical property of thechannel along at least a portion of the length of the channel prior tointroduction of the sample into the channel; comparing the measuredelectrical property value and the reference electrical property value;and determining whether a nucleic acid is present in the channel basedon the comparison between the measured electrical property value and thereference electrical property value.
 2. The method of claim 1, furthercomprising: prior to introducing the sample into the channel, measuringone or more electrical properties of the channel along at least theportion of the length of the channel; and determining the referenceelectrical property value based on the one or more electrical propertiesof the channel measured during the previous measuring step.
 3. Themethod of claim 1, further comprising: modifying at least a portion ofan inner surface of the channel with a material that covalently attachesto a nucleic acid probe; and covalently attaching the nucleic acid probeto the at least a portion of the inner surface of the channel.
 4. Themethod of claim 1, further comprising: introducing a nucleic acid probeinto the channel prior to measurement of the electrical property value.5. The method of claim 1, further comprising: applying a potentialdifference across the length of the channel after introducing the sampleinto the channel and prior to detecting the measured electrical propertyvalue.
 6. The method of claim 1, further comprising: applying apotential difference across the length of the channel after introducinga nucleic acid probe into the channel and prior to detecting thereference electrical property value.
 7. The method of claim 1, furthercomprising: displaying, on a visual display device, an indication ofwhether the nucleic acid is present in the sample.
 8. The method ofclaim 1, wherein the electrical property value corresponds to a value ofan electrical current conducted along at least the portion of length ofthe channel or an electrical conductance along at least the portion ofthe length of the channel.
 9. The method of claim 1, wherein the channelis configured to have a length ranging from 10 nanometers to 10centimeters.
 10. The method of claim 1, wherein the channel isconfigured to have a width ranging from 1 nanometer to 50 microns. 11.The method of claim 1, wherein the channel is configured to have a depthranging from 1 nanometer to 1 micron.
 12. The method of claim 1, furthercomprising: determining whether an additional analyte is present in thechannel based on the difference between the measured electrical propertyvalue and a second reference electrical property value.
 13. The methodof claim 1, further comprising: monitoring a first set of one or morevalues of the electrical property in the channel during a first timeperiod, and a second set of one or more values of the electricalproperty in the channel during a second time period; selecting thereference electrical property value from the first set of values uponequilibration of the one or more values in the channel during the firsttime period; and selecting the measured electrical property value fromthe second set of values upon equilibration of the one or more values inthe channel during the second time period.
 14. The method of claim 1,further comprising: determining, based on the measured electricalproperty value, a concentration of the nucleic acid in the sample. 15.The method of claim 1, further comprising: determining, based on themeasured electrical property value, a concentration of the nucleic acidin the sample relative to a concentration of an additional analyte inthe sample.
 16. The method of claim 1, further comprising: preparing thechannel for reuse by introducing a de-aggregation agent into thechannel, the de-aggregation agent causing disintegration of an aggregateformed in the channel by an interaction between the nucleic acid and anucleic acid probe.
 17. A method for detecting the presence or absenceof a nucleic acid in a sample, the method comprising: introducing anucleic acid probe into a channel, the channel having a length and awidth, the length being substantially greater than the width; measuringone or more electrical properties of the channel along at least aportion of the length of the channel; determining a reference channelelectrical property value based on the one or more electrical propertiesof the channel measured during the previous measuring step; introducinga sample into the channel; measuring the one or more electricalproperties of the channel along at least the portion of the length ofthe channel after the sample and the nucleic acid probe are introducedinto the channel; determining an electrical property value based on theone or more electrical properties measured after the nucleic acid probeand the sample are introduced into the channel; determining anydifferences between the reference channel electrical property value andthe electrical property value; and determining whether a nucleic acid ispresent in the channel based on the differences, if any, between thereference channel electrical property value and the electrical propertyvalue.
 18. A method for detecting the presence or absence of a nucleicacid in a sample, the method comprising: introducing a sample into achannel, the channel having a length and a width, the length beingsubstantially greater than the width; measuring one or more electricalproperties of the channel along at least a portion of the length of thechannel; determining a reference channel electrical property value basedon the one or more electrical properties of the channel measured duringthe previous measuring step; introducing one or more nucleic acid probesinto the channel; measuring the one or more electrical properties of thechannel along at least the portion of the length of the channel afterthe sample and the one or more nucleic acid probes are introduced intothe channel; determining an electrical property value based on the oneor more electrical properties measured after the sample and the one ormore nucleic acid probes are introduced into the channel; determiningany differences between the reference channel electrical property valueand the electrical property value; and determining whether the nucleicacid is present in the channel based on the differences, if any, betweenthe reference channel electrical property value and the electricalproperty value.
 19. A method for detecting the presence or absence of anucleic acid in a sample, the method comprising: coating at least aportion of an inner surface of a channel with a nucleic acid probe, thechannel having a length and a width, the length substantially greaterthan the width; measuring one or more electrical properties of thechannel along at least a portion of the length of the channel after thechannel is coated with the nucleic acid probe; determining a referencechannel electrical property value based on the one or more electricalproperties of the channel measured during the previous measuring step;and storing the reference channel electrical property value for use indetermining whether or not a nucleic acid is present in a sampleintroduced in the channel.
 20. A method for detecting the presence orabsence of a nucleic acid in a sample, the method comprising:introducing a sample and one or more nucleic acid probes into a channel,the channel having a length and a width, the length substantiallygreater than the width; applying a first potential difference across thelength of the channel in a first direction along the length of thechannel; measuring a first electrical property value of an electricalproperty along at least a portion of the length of the channel while thefirst potential difference is applied; applying a second potentialdifference across the length of the channel in a second direction alongthe length of the channel, the second direction opposite to the firstdirection; measuring a second electrical property value of theelectrical property along at least the portion of the length of thechannel while the second potential difference is applied; comparing thefirst and second electrical property values; and determining whether anucleic acid is present in the channel based on the comparison betweenthe first and second electrical property values.
 21. The method of claim20, wherein presence of the nucleic acid is detected if the first andsecond electrical property values are substantially unequal.
 22. Themethod of claim 20, wherein absence of the nucleic acid is detected ifthe first and second electrical property values are substantially equal.23. The method of claim 20, wherein the sample and the one or morenucleic acid probes are introduced into the channel concurrently. 24.The method of claim 20, wherein the sample and the one or more nucleicacid probes are introduced into the channel sequentially.
 25. The methodof claim 20, wherein an inner surface of the channel is modified tocovalently bind to at least one of the one or more nucleic acid probes.26. The method of claim 20, wherein the first and second electricalproperty values correspond to electrical current values conducted alongat least the portion of length of the channel or to electricalconductivity values along at least the portion of the length of thechannel.
 27. A nucleic acid detection system, comprising: a substrate,the substrate having at least one channel, the at least one channelhaving a length and a width, the length substantially greater than thewidth; a first port in fluid communication with a first end section ofthe at least one channel; a second port in fluid communication with asecond end section of the at least one channel; a first electrodeelectrically connected at the first end section of the at least onechannel and a second electrode electrically connected at the second endsection of the at least one channel, the first and second electrodeselectrically connected to their respective first and second end sectionsof the at least one channel to form a channel circuit, the channelcircuit having electrical properties and configured such that when anelectrically conductive fluid is present in the at least one channel,the electrically conductive fluid alters the electrical properties ofthe channel circuit; and a nucleic acid detection circuit in electricalcommunication with the first and second electrodes, the nucleic aciddetection circuit including a measurement circuit in electricalcommunication with the first and second electrode, the measurementcircuit having a measurement circuit output, the measurement circuitoutput including one or more values indicative of one or more electricalproperties of the channel circuit, the nucleic acid detection circuitincluding a memory in electrical communication with the measurementcircuit output and configured to store the one or more values indicativeof the one or more electrical properties of the channel circuitincluding at least a first value of an electrical property of thechannel circuit and a second value of the electrical property of thechannel circuit, the nucleic acid detection circuit further including acomparison circuit in electrical communication with the memory andhaving as inputs the at least first and second values, the comparisoncircuit configured to provide a comparison circuit output based at leastin part on the at least first and/or second values, the comparisoncircuit output indicative of whether a nucleic acid is present in the atleast one channel.
 28. The nucleic acid detection system of claim 27,wherein at least a portion of an inner surface of the at least onechannel is modified with a material that covalently attaches to anucleic acid probe.
 29. A nucleic acid detection system, comprising:means for accommodating a fluid flow; means for introducing a fluid at afirst terminal end of the means for accommodating the fluid flow; meansfor outputting the fluid at a second terminal end of the means foraccommodating the fluid flow; means for detecting first and secondvalues of an electrical property of the fluid between the first andsecond terminal ends of the means for accommodating the fluid flow; andmeans for determining whether a nucleic acid is present in the fluidbased on a difference between the first and second values of theelectrical property.